Terminally modified RNA

ABSTRACT

The invention relates to compositions and methods for the manufacture and optimization of modified mRNA molecules via optimization of their terminal architecture.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/729,933, filed Nov. 26, 2012, entitled Terminally OptimizedModified RNAs, U.S. Provisional Patent Application No. 61/737,224, filedDec. 14, 2012, entitled Terminally Optimized Modified RNAs, U.S.Provisional Patent Application No. 61/758,921, filed Jan. 31, 2013,entitled Differential Targeting Using RNA Constructs, U.S. ProvisionalPatent Application No. 61/781,139, filed Mar. 14, 2013, entitledDifferential Targeting Using RNA Constructs, U.S. Provisional PatentApplication No. 61/829,359, filed May 31, 2013, entitled DifferentialTargeting Using RNA Constructs, U.S. Provisional Patent Application No.61/839,903, filed Jun. 27, 2013, entitled Differential Targeting UsingRNA Constructs, U.S. Provisional Patent Application No. 61/842,709,filed Jul. 3, 2013, entitled Differential Targeting Using RNAConstructs, U.S. Provisional Patent Application No. 61/857,436, filedJul. 23, 2013, entitled Differential Targeting Using RNA Constructs,U.S. Provisional Patent Application No. 61/775,509, filed Mar. 9, 2013,entitled Heterologous Untranslated Regions for mRNA and U.S. ProvisionalPatent Application No. 61/829,372, filed May 31, 2013, entitledHeterologous Untranslated Regions for mRNA; the contents of each ofwhich are herein incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledM39US2.txt created on Oct. 1, 2013 which is 3,263,045 bytes in size. Theinformation in electronic format of the sequence listing is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to compositions and methods for the manufactureand use of modified and/or optimized mRNA and their use in combinationwith one or more modified or wild type mRNA encoding an RNA bindingprotein.

BACKGROUND OF THE INVENTION

Naturally occurring RNAs are synthesized from four basicribonucleotides: ATP, CTP, UTP and GTP, but may containpost-transcriptionally modified nucleotides. Further, approximately onehundred different nucleoside modifications have been identified in RNA(Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA ModificationDatabase: 1999 update. Nucl Acids Res 27: 196-197, herein incorporatedby reference in its entirety).

There are multiple problems with prior methodologies of effectingprotein expression. For example, heterologous deoxyribonucleic acid(DNA) introduced into a cell can be inherited by daughter cells (whetheror not the heterologous DNA has integrated into the chromosome) or byoffspring. Introduced DNA can integrate into host cell genomic DNA atsome frequency, resulting in alterations and/or damage to the host cellgenomic DNA. In addition, multiple steps must occur before a protein ismade. Once inside the cell, DNA must be transported into the nucleuswhere it is transcribed into RNA. The RNA transcribed from DNA must thenenter the cytoplasm where it is translated into protein. This need formultiple processing steps creates lag times before the generation of aprotein of interest. Further, it is difficult to obtain DNA expressionin cells; frequently DNA enters cells but is not expressed or notexpressed at reasonable rates or concentrations. This can be aparticular problem when DNA is introduced into cells such as primarycells or modified cell lines. The role of nucleoside modifications onthe immuno-stimulatory potential, stability, and on the translationefficiency of RNA, and the consequent benefits to this for enhancingprotein expression and producing therapeutics have been previouslyexplored. Such studies are detailed in published co-pendingInternational Publication No WO2012019168 filed Aug. 5, 2011,International Publication No WO2012045082 filed Oct. 3, 2011,International Publication No WO2012045075 filed Oct. 3, 2011,International Publication No WO2013052523 filed Oct. 3, 2012, andInternational Publication No WO2013090648 filed Dec. 14, 2012 thecontents of which are incorporated herein by reference in theirentirety.

The use of modified polynucleotides in the fields of antibodies,viruses, veterinary applications and a variety of in vivo settings havebeen explored and are disclosed in, for example, co-pending and co-ownedU.S. Provisional Patent Application No. 61/618,862, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Biologics; U.S.Provisional Patent Application No. 61/681,645, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Biologics; U.S.Provisional Patent Application No. 61/737,130, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Biologics; U.S.Provisional Patent Application No. 61/618,866, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Antibodies; U.S.Provisional Patent Application No. 61/681,647, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Antibodies; U.S.Provisional Patent Application No. 61/737,134, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Antibodies; U.S.Provisional Patent Application No. 61/618,868, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Vaccines; U.S.Provisional Patent Application No. 61/681,648, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Vaccines; U.S.Provisional Patent Application No. 61/737,135, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Vaccines; U.S.Provisional Patent Application No. 61/618,870, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of TherapeuticProteins and Peptides; U.S. Provisional Patent Application No.61/681,649, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Therapeutic Proteins and Peptides; U.S. ProvisionalPatent Application No. 61/737,139, filed Dec. 14, 2012, ModifiedPolynucleotides for the Production of Therapeutic Proteins and Peptides;U.S. Provisional Patent Application No. 61/618,873, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of SecretedProteins; U.S. Provisional Patent Application No. 61/681,650, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins; U.S. Provisional Patent Application No. 61/737,147,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Secreted Proteins; U.S. Provisional Patent Application No.61/618,878, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Plasma Membrane Proteins; U.S. Provisional PatentApplication No. 61/681,654, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Plasma Membrane Proteins; U.S.Provisional Patent Application No. 61/737,152, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins; U.S. Provisional Patent Application No. 61/618,885, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins; U.S. Provisional PatentApplication No. 61/681,658, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins; U.S. Provisional Patent Application No. 61/737,155, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins; U.S. Provisional PatentApplication No. 61/618,896, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins; U.S. Provisional Patent Application No. 61/668,157, filed Jul.5, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins; U.S. Provisional PatentApplication No. 61/681,661, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins; U.S. Provisional Patent Application No. 61/737,160, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins; U.S. Provisional PatentApplication No. 61/618,911, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Nuclear Proteins; U.S. ProvisionalPatent Application No. 61/681,667, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Nuclear Proteins; U.S.Provisional Patent Application No. 61/737,168, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins; U.S. Provisional Patent Application No. 61/618,922, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins; U.S. Provisional Patent Application No. 61/681,675, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins; U.S. Provisional Patent Application No. 61/737,174, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins; U.S. Provisional Patent Application No. 61/618,935, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/681,687, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/737,184, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/618,945, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/681,696, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/737,191, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/618,953, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/681,704, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/737,203, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/681,720, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Cosmetic Proteins and Peptides;U.S. Provisional Patent Application No. 61/737,213, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of CosmeticProteins and Peptides; U.S. Provisional Patent Application No.61/681,742, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Oncology-Related Proteins and Peptides; InternationalApplication No PCT/US2013/030062, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Biologics and Proteins Associatedwith Human Disease; U.S. patent application Ser. No. 13/791,922, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofBiologics and Proteins Associated with Human Disease; InternationalApplication No PCT/US2013/030063, filed Mar. 9, 2013, entitled ModifiedPolynucleotides; International Application No. PCT/US2013/030064,entitled Modified Polynucleotides for the Production of SecretedProteins; U.S. patent application Ser. No. 13/791,921, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of SecretedProteins; International Application No PCT/US2013/030059, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of MembraneProteins; International Application No. PCT/US2013/030066, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins; International Application No.PCT/US2013/030067, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Nuclear Proteins; International Application No.PCT/US2013/030060, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Proteins; International Application No.PCT/US2013/030061, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Proteins Associated with Human Disease; U.S.patent application Ser. No. 13/791,910, filed Mar. 9, 2013, entitledModified Polynucleotides for the Production of Proteins Associated withHuman Disease; International Application No. PCT/US2013/030068, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofCosmetic Proteins and Peptides; and International Application No.PCT/US2013/030070, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Oncology-Related Proteins and Peptides;International Patent Application No. PCT/US2013/031821, filed Mar. 15,2013, entitled In Vivo Production of Proteins; the contents of each ofwhich are herein incorporated by reference in their entireties.

Formulations and delivery of modified polynucleotides are described in,for example, co-pending and co-owned International Publication NoWO2013090648, filed Dec. 14, 2012, entitled Modified Nucleoside,Nucleotide, Nucleic Acid Compositions and US Publication NoUS20130156849, filed Dec. 14, 2012, entitled Modified Nucleoside,Nucleotide, Nucleic Acid Compositions; the contents of each of which areherein incorporated by reference in their entireties.

There is a need in the art, therefore, for biological modalities toaddress the modulation of intracellular translation of nucleic acids.The present invention addresses this need by providing methods andcompositions for the manufacture and optimization of modified mRNAmolecules via alteration of the terminal architecture of the molecules.

SUMMARY OF THE INVENTION

Disclosed herein are methods of stabilizing or inducing increasedprotein expression from a modified mRNA. In another method, a cell iscontacted with a modified mRNA encoding a polypeptide of interest incombination with a modified mRNA encoding one or more RNA bindingproteins.

In one embodiment, provided herein are terminally optimized mRNAcomprising first region of linked nucleosides encoding a polypeptide ofinterest which is located 5′ relative to the first region, a secondterminal region located 3′ relative to the first terminal region and a3′ tailing region. The first terminal region may comprise at least onetranslation enhancer element (TEE) such as, but not limited to, the TEEsdescribed in Table 28 such as, but not limited to, TEE-001-TEE-705.

The first terminal region may comprise a 5′ untranslated region (UTR)which may be the native 5′UTR of the encoded polypeptide of interest ormay be heterologous to the encoded polypeptide of interest. In oneaspect, the 5′UTR may comprise at least one translation initiationsequence such as a kozak sequence, an internal ribosome entry site(IRES) and/or a fragment thereof. As a non-limiting example, the 5′UTRmay comprise at least one fragment of an IRES. As another non-limitingexample, the 5′UTR may comprise at least 5 fragments of an IRES. Inanother aspect, the 5′UTR may comprise a structured UTR.

The second terminal region may comprise at least one microRNA bindingsite, seed sequence or microRNA binding site without a seed sequence. Inone aspect, the microRNA is an immune cell specific microRNA such as,but not limited to, mir-122, miR-142-3p, miR-142-5p, miR-146a andmiR-146b.

In one embodiment, the 3′ tailing region may comprise a chainterminating nucleoside such as, but not limited to, 3′-deoxyadenosine(cordycepin), 3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine,3′-deoxythymine, 2′,3′-dideoxynucleosides, 2′,3′-dideoxyadenosine,2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine,2′,3′-dideoxythymine, a 2′-deoxynucleoside, and —O— methylnucleoside. Inone aspect, the 3′ tailing region is a stem loop sequence or a polyAtail.

In one embodiment, provided herein are terminally optimized mRNAcomprising first region of linked nucleosides encoding a polypeptide ofinterest which is located 5′ relative to the first region, a secondterminal region located 3′ relative to the first terminal region and a3′ tailing region of linked nucleosides and at least one chainterminating nucleoside located 3′ relative to the terminally optimizedmRNA. In one aspect, the second terminal region may comprise at leastone microRNA binding site, seed sequence or microRNA binding sitewithout a seed sequence. In one aspect, the microRNA is an immune cellspecific microRNA such as, but not limited to, mir-122, miR-142-3p,miR-142-5p, miR-146a and miR-146b.

The terminally optimized mRNA described herein may comprise at least onemodified nucleoside. In one embodiment, the terminally optimized mRNAcomprises a pseudouridine analog such as, but not limited to,1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine,1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine,1-methyl-pseudouridine (m¹ψ), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine,1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ), and2′-O-methyl-pseudouridine (ψm). In another embodiment, the terminallyoptimized mRNA comprises the pseudouridine analog 1-methylpseudouridine.In yet another embodiment, the terminally optimized mRNA comprises thepseudouridine analog 1-methylpseudouridine and comprises the modifiednucleoside 5-methylcytidine.

The terminally optimized mRNA described herein may comprise at least one5′ cap structure such as, but not limited to, Cap0, Cap1, ARCA, inosine,N1-methyl-guanosine, 2′ fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine,Cap2, Cap4, and CAP-003-CAP-225.

In one aspect, at least one region of the terminally optimized mRNA maybe codon optimized. As a non-limiting example, the first region oflinked nucleosides may be codon optimized.

Also provided herein are methods of using the terminally optimized mRNA.

In one embodiment, provided is a method of reducing antigen-mediatedimmune response in an organism by contacting the organism with aterminally optimized mRNA. The terminally optimized mRNA may comprise afirst region of linked nucleosides encoding a polypeptide of interestwhich is located 5′ relative to the first region, a second terminalregion located 3′ relative to the first terminal region and a 3′tailingregion. The second terminal region may comprise at least one microRNAbinding site, seed sequence or microRNA binding site without a seedsequence. In one aspect, the microRNA is an immune cell specificmicroRNA such as, but not limited to, mir-122, miR-142-3p, miR-142-5p,miR-146a and miR-146b.

In a another embodiment, terminally optimized mRNA which reduces theantigen-mediated immune response may comprise at least one translationenhancer element (TEE) sequence such as, but not limited to, TEE-001-TEE705, a chain terminating nucleoside and/or a stem loop sequence.

In yet another embodiment, terminally optimized mRNA which reduces theantigen-mediated immune response may comprise at least one region whichis codon optimized. As a non-limiting example, the first region oflinked nucleosides may be codon optimized.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a primary construct of the present invention.

FIG. 2 is an expanded schematic of the second flanking region of aprimary construct of the present invention illustrating the sensorelements of the polynucleotide.

FIG. 3 is a clone map useful in the present invention.

FIG. 4 is a histogram showing the improved protein production frommodified mRNAs of the present invention having increasingly longerpoly-A tails at two concentrations.

DETAILED DESCRIPTION

Described herein are compositions and methods for the manufacture andoptimization of modified mRNA molecules via alteration of the terminalarchitecture of the molecules. Specifically disclosed are methods forincreasing protein production by altering the terminal regions of themRNA. Such terminal regions include at least the 5′ untranslated region(UTR), and 3′UTR. Other features which may be modified and found to the5′ or 3′ of the coding region include the 5′ cap and poly-A tail of themodified mRNAs (modified RNAs).

In general, exogenous nucleic acids, particularly viral nucleic acids,introduced into cells induce an innate immune response, resulting ininterferon (IFN) production and cell death. However, it is of greatinterest for therapeutics, diagnostics, reagents and for biologicalassays to deliver a nucleic acid, e.g., a ribonucleic acid (RNA) insidea cell, either in vivo or ex vivo, such as to cause intracellulartranslation of the nucleic acid and production of the encoded protein.Of particular importance is the delivery and function of anon-integrative nucleic acid, as nucleic acids characterized byintegration into a target cell are generally imprecise in theirexpression levels, deleteriously transferable to progeny and neighborcells, and suffer from the substantial risk of mutation.

The terminal modification described herein may be used in the modifiednucleic acids encoding polypeptides of interest, such as, but notlimited to, the polypeptides of interest described in, U.S. ProvisionalPatent Application No. 61/618,862, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Biologics, U.S. Provisional PatentApplication No. 61/681,645, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Biologics, U.S. Provisional PatentApplication No. 61/737,130, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Biologics, U.S. Provisional PatentApplication No. 61/618,866, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Antibodies, U.S. ProvisionalPatent Application No. 61/681,647, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Antibodies, U.S.Provisional Patent Application No. 61/737,134, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Antibodies, U.S.Provisional Patent Application No. 61/618,868, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Vaccines, U.S.Provisional Patent Application No. 61/681,648, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Vaccines, U.S.Provisional Patent Application No. 61/737,135, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Vaccines, U.S.Provisional Patent Application No. 61/618,870, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of TherapeuticProteins and Peptides, U.S. Provisional Patent Application No.61/681,649, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Therapeutic Proteins and Peptides, U.S. ProvisionalPatent Application No. 61/737,139, filed Dec. 14, 2012, ModifiedPolynucleotides for the Production of Therapeutic Proteins and Peptides,U.S. Provisional Patent Application No. 61/618,873, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of SecretedProteins, U.S. Provisional Patent Application No. 61/681,650, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins, U.S. Provisional Patent Application No. 61/737,147,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Secreted Proteins, U.S. Provisional Patent Application No.61/618,878, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Plasma Membrane Proteins, U.S. Provisional PatentApplication No. 61/681,654, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Plasma Membrane Proteins, U.S.Provisional Patent Application No. 61/737,152, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins, U.S. Provisional Patent Application No. 61/618,885, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/681,658, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins, U.S. Provisional Patent Application No. 61/737,155, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/618,896, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins, U.S. Provisional Patent Application No. 61/668,157, filed Jul.5, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/681,661, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins, U.S. Provisional Patent Application No. 61/737,160, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/618,911, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Nuclear Proteins, U.S. ProvisionalPatent Application No. 61/681,667, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Nuclear Proteins, U.S.Provisional Patent Application No. 61/737,168, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins, U.S. Provisional Patent Application No. 61/618,922, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/681,675, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/737,174, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/618,935, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/681,687, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/737,184, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/618,945, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/681,696, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/737,191, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Patent Application No. 61/618,953, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of ProteinsAssociated with Human Disease, U.S. Patent Application No. 61/681,704,filed Aug. 10, 2012, entitled Modified Polynucleotides for theProduction of Proteins Associated with Human Disease, U.S. PatentApplication No. 61/737,203, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, International Application No PCT/US2013/030062, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of Biologicsand Proteins Associated with Human Disease; International Application NoPCT/US2013/030063, filed Mar. 9, 2013, entitled ModifiedPolynucleotides; International Application No. PCT/US2013/030064,entitled Modified Polynucleotides for the Production of SecretedProteins; International Application No PCT/US2013/030059, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of MembraneProteins; International Application No. PCT/US2013/030066, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins; International Application No.PCT/US2013/030067, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Nuclear Proteins; International Application No.PCT/US2013/030060, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Proteins; International Application No.PCT/US2013/030061, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Proteins Associated with Human Disease;International Application No. PCT/US2013/030068, filed Mar. 9, 2013,entitled Modified Polynucleotides for the Production of CosmeticProteins and Peptides; International Application No. PCT/US2013/030070,filed Mar. 9, 2013, entitled Modified Polynucleotides for the Productionof Oncology-Related Proteins and Peptides; and International ApplicationNo. PCT/US2013/031821, filed Mar. 15, 2013, entitled In Vivo Productionof Proteins, U.S. Provisional Patent Application No. 61/753,661, filedJan. 17, 2013, entitled Signal-Sensor Polynucleotide for the Alterationof Cellular Phenotypes and Microenvironments, U.S. Provisional PatentApplication No. 61/754,159, filed Jan. 18, 2013, entitled Signal-SensorPolynucleotide for the Alteration of Cellular Phenotypes andMicroenvironments, U.S. Provisional Patent Application No. 61/781,097,filed Mar. 14, 2013, entitled Signal-Sensor Polynucleotide for theAlteration of Cellular Phenotypes and Microenvironments, U.S.Provisional Patent Application No. 61/829,334, filed May 31, 2013,entitled Signal-Sensor Polynucleotide for the Alteration of CellularPhenotypes and Microenvironments, U.S. Provisional Patent ApplicationNo. 61/729,933, filed Nov. 26, 2012, entitled Terminally OptimizedModified RNAs, U.S. Provisional Patent Application No. 61/737,224, filedDec. 14, 2012, entitled Terminally Optimized Modified RNAs, U.S.Provisional Patent Application No. 61/758,921, filed Jan. 31, 2013,entitled Differential Targeting Using RNA Constructs, U.S. ProvisionalPatent Application No. 61/781,139, filed Mar. 14, 2013, entitledDifferential Targeting Using RNA Constructs, U.S. ProvisionalApplication No. 61/829,359, filed May 31, 2013, entitled DifferentialTargeting Using RNA Constructs, the contents of each of which are hereinincorporated by reference in their entireties.

Provided herein in part are nucleic acid molecules encoding polypeptidescapable of modulating a cell's status, function and/or activity, andmethods of making and using these nucleic acids and polypeptides. Asdescribed herein and in co-pending and co-owned InternationalPublication No WO2012019168 filed Aug. 5, 2011, InternationalPublication No WO2012045082 filed Oct. 3, 2011, InternationalPublication No WO2012045075 filed Oct. 3, 2011, InternationalPublication No WO2013052523 filed Oct. 3, 2012, and InternationalPublication No WO2013090648 filed Dec. 14, 2012, the contents of each ofwhich are incorporated by reference herein in their entirety, thesemodified nucleic acid molecules are capable of reducing the innateimmune activity of a population of cells into which they are introduced,thus increasing the efficiency of protein production in that cellpopulation.

In addition to utilization of non-natural nucleosides and nucleotides,such as those described in US Patent Publication No US20130115272, filedOct. 3, 2012 (the contents of which are herein incorporated by referencein its entirety), in the modified RNAs of the present invention, it hasnow been discovered that concomitant use of altered terminalarchitecture may also serve to increase protein production from a cellpopulation.

I. Compositions of the Invention

This invention provides nucleic acid molecules, including RNAs such asmRNAs, which may be synthetic, that contain one or more modifiednucleosides (termed “modified nucleic acids” or “modified nucleic acidmolecules”) and polynucleotides, primary constructs and modified mRNA(mmRNA), which have useful properties including the lack of asubstantial induction of the innate immune response of a cell into whichthe mRNA is introduced. Because these modified nucleic acids enhance theefficiency of protein production, intracellular retention of nucleicacids, and viability of contacted cells, as well as possess reducedimmunogenicity, these nucleic acids having these properties are termed“enhanced” nucleic acids or modified RNAs herein.

In one embodiment, the polynucleotides are nucleic acid transcriptswhich encode one or more polypeptides of interest that, when translated,deliver a signal to the cell which results in the therapeutic benefit tothe organism. The signal polynucleotides may optionally further comprisea sequence (translatable or not) which sense the microenvironment of thepolynucleotide and alters (a) the function or phenotype outcomeassociated with the peptide or protein which is translated, (b) theexpression level of the signal polynucleotide, and/or both.

The term “nucleic acid,” in its broadest sense, includes any compoundand/or substance that comprise a polymer of nucleotides. These polymersare often referred to as polynucleotides.

Exemplary nucleic acids include ribonucleic acids (RNAs),deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycolnucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids(LNAs) or hybrids thereof. They may also include RNAi-inducing agents,RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes,catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers,vectors, etc. In preferred embodiments, the modified nucleic acidmolecule is one or more messenger RNAs (mRNAs).

In preferred embodiments, the polynucleotide or nucleic acid molecule isa messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA)refers to any polynucleotide which encodes a polypeptide of interest andwhich is capable of being translated to produce the encoded polypeptideof interest in vitro, in vivo, in situ or ex vivo. Polynucleotides ofthe invention may be mRNA or any nucleic acid molecule and may or maynot be chemically modified.

Traditionally, the basic components of an mRNA molecule include at leasta coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. Buildingon this wild type modular structure, the present invention expands thescope of functionality of traditional mRNA molecules by providingpolynucleotides or primary RNA constructs which maintain a modularorganization, but which comprise one or more structural and/or chemicalmodifications or alterations which impart useful properties to thepolynucleotide including, in some embodiments, the lack of a substantialinduction of the innate immune response of a cell into which thepolynucleotide is introduced. As such, modified mRNA molecules of thepresent invention, which may be synthetic, are termed “mmRNA.” As usedherein, a “structural” feature or modification is one in which two ormore linked nucleotides are inserted, deleted, duplicated, inverted orrandomized in a polynucleotide polynucleotide, primary construct ormmRNA without significant chemical modification to the nucleotidesthemselves. Because chemical bonds will necessarily be broken andreformed to effect a structural modification, structural modificationsare of a chemical nature and hence are chemical modifications. However,structural modifications will result in a different sequence ofnucleotides. For example, the polynucleotide “ATCG” may be chemicallymodified to “AT-5meC-G”. The same polynucleotide may be structurallymodified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has beeninserted, resulting in a structural modification to the polynucleotide.

Provided are modified nucleic acids containing a translatable region andone, two, or more than two different nucleoside modifications. In someembodiments, the modified nucleic acid exhibits reduced degradation in acell into which the nucleic acid is introduced, relative to acorresponding unmodified nucleic acid.

In some embodiments, the chemical modifications can be located on thesugar moiety of the nucleotide

In some embodiments, the chemical modifications can be located on thephosphate backbone of the nucleotide

In certain embodiments it is desirable to intracellularly degrade amodified nucleic acid introduced into the cell, for example if precisetiming of protein production is desired. Thus, the invention provides amodified nucleic acid containing a degradation domain, which is capableof being acted on in a directed manner within a cell.

Polynucleotide, Primary Construct or mmRNA Architecture

The polynucleotides of the present invention are distinguished from wildtype mRNA in their functional and/or structural design features whichserve to, as evidenced herein, overcome existing problems of effectivepolypeptide production using nucleic acid-based therapeutics.

FIG. 1 shows a representative primary construct 100 of the presentinvention. As used herein, the term “primary construct” or “primary mRNAconstruct” refers to polynucleotide transcript which encodes one or morepolypeptides of interest and which retains sufficient structural and/orchemical features to allow the polypeptide of interest encoded thereinto be translated. Primary constructs may be polynucleotides of theinvention. When structurally or chemically modified, the primaryconstruct may be referred to as a mmRNA.

Returning to FIG. 1, the primary construct 100 here contains a firstregion of linked nucleotides 102 that is flanked by a first flankingregion 104 and a second flaking region 106. As used herein, the “firstregion” may be referred to as a “coding region” or “region encoding” orsimply the “first region.” This first region may include, but is notlimited to, the encoded polypeptide of interest. The polypeptide ofinterest may comprise at its 5′ terminus one or more signal peptidesequences encoded by a signal peptide sequence region 103. The flankingregion 104 may comprise a region of linked nucleotides comprising one ormore complete or incomplete 5′ UTRs sequences. The flanking region 104may also comprise a 5′ terminal cap 108. The second flanking region 106may comprise a region of linked nucleotides comprising one or morecomplete or incomplete 3′ UTRs. The flanking region 106 may alsocomprise a 3′ tailing sequence 110 and a 3′UTR 120.

Bridging the 5′ terminus of the first region 102 and the first flankingregion 104 is a first operational region 105. Traditionally thisoperational region comprises a start codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a start codon.

Bridging the 3′ terminus of the first region 102 and the second flankingregion 106 is a second operational region 107. Traditionally thisoperational region comprises a stop codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a stop codon. According to the present invention, multipleserial stop codons may also be used. In one embodiment, the operationregion of the present invention may comprise two stop codons. The firststop codon may be “TGA” and the second stop codon may be selected fromthe group consisting of “TAA,” “TGA” and “TAG.” The operation region mayfurther comprise three stop codons. The third stop codon may be selectedfrom the group consisting of “TAA,” “TGA” and “TAG.”

Turning to FIG. 2, the 3′UTR 120 of the second flanking region 106 maycomprise one or more sensor sequences 130. These sensor sequences asdiscussed herein operate as pseudo-receptors (or binding sites) forligands of the local microenvironment of the primary construct orpolynucleotide. For example, microRNA binding sites or miRNA seeds maybe used as sensors such that they function as pseudoreceptors for anymicroRNAs present in the environment of the polynucleotide.

Generally, the shortest length of the first region of the primaryconstruct of the present invention can be the length of a nucleic acidsequence that is sufficient to encode for a dipeptide, a tripeptide, atetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, anoctapeptide, a nonapeptide, or a decapeptide. In another embodiment, thelength may be sufficient to encode a peptide of 2-30 amino acids, e.g.5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may besufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17,20, 25 or 30 amino acids, or a peptide that is no longer than 40 aminoacids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10amino acids. Examples of dipeptides that the polynucleotide sequencescan encode or include, but are not limited to, carnosine and anserine.

Generally, the length of the first region encoding the polypeptide ofinterest of the present invention is greater than about 30 nucleotidesin length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000,7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). Asused herein, the “first region” may be referred to as a “coding region”or “region encoding” or simply the “first region.”

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes from about 30 to about 100,000 nucleotides (e.g., from 30 to50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000,from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000,from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000,from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000,from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000,from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to70,000, and from 2,000 to 100,000).

According to the present invention, the first and second flankingregions may range independently from 15-1,000 nucleotides in length(e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,and 1,000 nucleotides).

According to the present invention, the tailing sequence may range fromabsent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Wherethe tailing region is a polyA tail, the length may be determined inunits of or as a function of polyA binding protein binding. In thisembodiment, the polyA tail is long enough to bind at least 4 monomers ofpolyA binding protein. PolyA binding protein monomers bind to stretchesof approximately 38 nucleotides. As such, it has been observed thatpolyA tails of about 80 nucleotides and 160 nucleotides are functional.

According to the present invention, the capping region may comprise asingle cap or a series of nucleotides forming the cap. In thisembodiment the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7,1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In someembodiments, the cap is absent.

According to the present invention, the first and second operationalregions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or30 or fewer nucleotides in length and may comprise, in addition to astart and/or stop codon, one or more signal and/or restrictionsequences.

Cyclic Polynucleotides

According to the present invention, a nucleic acid, modified RNA orprimary construct may be cyclized, or concatemerized, to generate atranslation competent molecule to assist interactions between poly-Abinding proteins and 5′-end binding proteins. The mechanism ofcyclization or concatemerization may occur through at least 3 differentroutes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newlyformed 5′-/3′-linkage may be intramolecular or intermolecular.

In the first route, the 5′-end and the 3′-end of the nucleic acidcontain chemically reactive groups that, when close together, form a newcovalent linkage between the 5′-end and the 3′-end of the molecule. The5′-end may contain an NHS-ester reactive group and the 3′-end maycontain a 3′-amino-terminated nucleotide such that in an organic solventthe 3′-amino-terminated nucleotide on the 3′-end of a synthetic mRNAmolecule will undergo a nucleophilic attack on the 5′-NHS-ester moietyforming a new 5′-/3′-amide bond.

In the second route, T4 RNA ligase may be used to enzymatically link a5′-phosphorylated nucleic acid molecule to the 3′-hydroxyl group of anucleic acid forming a new phosphorodiester linkage. In an examplereaction, 1 μg of a nucleic acid molecule is incubated at 37° C. for 1hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich,Mass.) according to the manufacturer's protocol. The ligation reactionmay occur in the presence of a split oligonucleotide capable ofbase-pairing with both the 5′- and 3′-region in juxtaposition to assistthe enzymatic ligation reaction.

In the third route, either the 5′- or 3′-end of the cDNA templateencodes a ligase ribozyme sequence such that during in vitrotranscription, the resultant nucleic acid molecule can contain an activeribozyme sequence capable of ligating the 5′-end of a nucleic acidmolecule to the 3′-end of a nucleic acid molecule. The ligase ribozymemay be derived from the Group I Intron, Group I Intron, Hepatitis DeltaVirus, Hairpin ribozyme or may be selected by SELEX (systematicevolution of ligands by exponential enrichment). The ribozyme ligasereaction may take 1 to 24 hours at temperatures between 0 and 37° C.

Polynucleotide Multimers

According to the present invention, multiple distinct nucleic acids,modified RNA or primary constructs may be linked together through the3′-end using nucleotides which are modified at the 3′-terminus. Chemicalconjugation may be used to control the stoichiometry of delivery intocells. For example, the glyoxylate cycle enzymes, isocitrate lyase andmalate synthase, may be supplied into HepG2 cells at a 1:1 ratio toalter cellular fatty acid metabolism. This ratio may be controlled bychemically linking nucleic acids or modified RNA using a 3′-azidoterminated nucleotide on one nucleic acids or modified RNA species and aC5-ethynyl or alkynyl-containing nucleotide on the opposite nucleicacids or modified RNA species. The modified nucleotide is addedpost-transcriptionally using terminal transferase (New England Biolabs,Ipswich, Mass.) according to the manufacturer's protocol. After theaddition of the 3′-modified nucleotide, the two nucleic acids ormodified RNA species may be combined in an aqueous solution, in thepresence or absence of copper, to form a new covalent linkage via aclick chemistry mechanism as described in the literature.

In another example, more than two polynucleotides may be linked togetherusing a functionalized linker molecule. For example, a functionalizedsaccharide molecule may be chemically modified to contain multiplechemical reactive groups (SH—, NH₂—, N₃, etc. . . . ) to react with thecognate moiety on a 3′-functionalized mRNA molecule (i.e., a3′-maleimide ester, 3′-NHS-ester, alkynyl). The number of reactivegroups on the modified saccharide can be controlled in a stoichiometricfashion to directly control the stoichiometric ratio of conjugatednucleic acid or mRNA.

Modified RNA Conjugates and Combinations

In order to further enhance protein production, nucleic acids, modifiedRNA, polynucleotides or primary constructs of the present invention canbe designed to be conjugated to other polynucleotides, dyes,intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases, proteins, e.g., glycoproteins, orpeptides, e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell, hormones and hormonereceptors, non-peptidic species, such as lipids, lectins, carbohydrates,vitamins, cofactors, or a drug.

Conjugation may result in increased stability and/or half life and maybe particularly useful in targeting the nucleic acids, modified RNA,polynucleotides or primary constructs to specific sites in the cell,tissue or organism.

According to the present invention, the nucleic acids, modified RNA orprimary construct may be administered with, or further encode one ormore of RNAi agents, siRNAs, shRNAs, miRNAs, miRNA binding sites,antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triplehelix formation, aptamers or vectors, and the like.

Bifunctional Polynucleotides

In one embodiment of the invention are bifunctional polynucleotides(e.g., bifunctional nucleic acids, bifunctional modified RNA orbifunctional primary constructs). As the name implies, bifunctionalpolynucleotides are those having or capable of at least two functions.These molecules may also by convention be referred to asmulti-functional.

The multiple functionalities of bifunctional polynucleotides may beencoded by the RNA (the function may not manifest until the encodedproduct is translated) or may be a property of the polynucleotideitself. It may be structural or chemical. Bifunctional modifiedpolynucleotides may comprise a function that is covalently orelectrostatically associated with the polynucleotides. Further, the twofunctions may be provided in the context of a complex of a modified RNAand another molecule.

Bifunctional polynucleotides may encode peptides which areanti-proliferative. These peptides may be linear, cyclic, constrained orrandom coil. They may function as aptamers, signaling molecules, ligandsor mimics or mimetics thereof. Anti-proliferative peptides may, astranslated, be from 3 to 50 amino acids in length. They may be 5-40,10-30, or approximately 15 amino acids long. They may be single chain,multichain or branched and may form complexes, aggregates or anymulti-unit structure once translated.

Noncoding Polynucleotides

As described herein, provided are nucleic acids, modified RNA,polynucleotides and primary constructs having sequences that arepartially or substantially not translatable, e.g., having a noncodingregion. Such molecules are generally not translated, but can exert aneffect on protein production by one or more of binding to andsequestering one or more translational machinery components such as aribosomal protein or a transfer RNA (tRNA), thereby effectively reducingprotein expression in the cell or modulating one or more pathways orcascades in a cell which in turn alters protein levels. The nucleicacids, polynucleotides, primary constructs or mRNA may contain or encodeone or more long noncoding RNA (lncRNA, or lincRNA) or portion thereof,a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interferingRNA (siRNA) or Piwi-interacting RNA (piRNA).

Polypeptides of Interest

According to the present invention, the primary construct is designed toencode one or more polypeptides of interest or fragments thereof. Apolypeptide of interest may include, but is not limited to, wholepolypeptides, a plurality of polypeptides or fragments of polypeptides,which independently may be encoded by one or more nucleic acids, aplurality of nucleic acids, fragments of nucleic acids or variants ofany of the aforementioned. As used herein, the term “polypeptides ofinterest” refers to any polypeptide which is selected to be encoded inthe primary construct of the present invention. As used herein,“polypeptide” means a polymer of amino acid residues (natural orunnatural) linked together most often by peptide bonds. The term, asused herein, refers to proteins, polypeptides, and peptides of any size,structure, or function. In some instances the polypeptide encoded issmaller than about 50 amino acids and the polypeptide is then termed apeptide. If the polypeptide is a peptide, it will be at least about 2,3, 4, or at least 5 amino acid residues long. Thus, polypeptides includegene products, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide may be a singlemolecule or may be a multi-molecular complex such as a dimer, trimer ortetramer. They may also comprise single chain or multichain polypeptidessuch as antibodies or insulin and may be associated or linked. Mostcommonly disulfide linkages are found in multichain polypeptides. Theterm polypeptide may also apply to amino acid polymers in which one ormore amino acid residues are an artificial chemical analogue of acorresponding naturally occurring amino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and preferably, they will be at least about 80%, morepreferably at least about 90% identical (homologous) to a native orreference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains one or more amino acids whichwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, e.g., phenylalanine may act as an inactivatingsubstitution for tyrosine; or alanine may act as an inactivatingsubstitution for serine.

“Homology” as it applies to amino acid sequences is defined as thepercentage of residues in the candidate amino acid sequence that areidentical with the residues in the amino acid sequence of a secondsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well known in the art. It is understoodthat homology depends on a calculation of percent identity but maydiffer in value due to gaps and penalties introduced in the calculation.

By “homologs” as it applies to polypeptide sequences means thecorresponding sequence of other species having substantial identity to asecond sequence of a second species.

“Analogs” is meant to include polypeptide variants which differ by oneor more amino acid alterations, e.g., substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present invention contemplates several types of compositions whichare polypeptide based including variants and derivatives. These includesubstitutional, insertional, deletion and covalent variants andderivatives. The term “derivative” is used synonymously with the term“variant” but generally refers to a molecule that has been modifiedand/or changed in any way relative to a reference molecule or startingmolecule.

As such, polynucleotides encoding polypeptides of interest containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences are included withinthe scope of this invention. For example, sequence tags or amino acids,such as one or more lysines, can be added to the peptide sequences ofthe invention (e.g., at the N-terminal or C-terminal ends). Sequencetags can be used for peptide purification or localization. Lysines canbe used to increase peptide solubility or to allow for biotinylation.Alternatively, amino acid residues located at the carboxy and aminoterminal regions of the amino acid sequence of a peptide or protein mayoptionally be deleted providing for truncated sequences. Certain aminoacids (e.g., C-terminal or N-terminal residues) may alternatively bedeleted depending on the use of the sequence, as for example, expressionof the sequence as part of a larger sequence which is soluble, or linkedto a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions may be single, where only one amino acid inthe molecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Insertional variants” when referring to polypeptides are those with oneor more amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved. Ordinarily, deletional variants will have one or more aminoacids deleted in a particular region of the molecule.

“Covalent derivatives” when referring to polypeptides includemodifications of a native or starting protein with an organicproteinaceous or non-proteinaceous derivatizing agent, and/orpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theprotein with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-proteinantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the polypeptides produced in accordancewith the present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

“Features” when referring to polypeptides are defined as distinct aminoacid sequence-based components of a molecule. Features of thepolypeptides encoded by the mmRNA of the present invention includesurface manifestations, local conformational shape, folds, loops,half-loops, domains, half-domains, sites, termini or any combinationthereof.

As used herein when referring to polypeptides the term “surfacemanifestation” refers to a polypeptide based component of a proteinappearing on an outermost surface.

As used herein when referring to polypeptides the term “localconformational shape” means a polypeptide based structural manifestationof a protein which is located within a definable space of the protein.

As used herein when referring to polypeptides the term “fold” refers tothe resultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of energetic forces.Regions formed in this way include hydrophobic and hydrophilic pockets,and the like.

As used herein the term “turn” as it relates to protein conformationmeans a bend which alters the direction of the backbone of a peptide orpolypeptide and may involve one, two, three or more amino acid residues.

As used herein when referring to polypeptides the term “loop” refers toa structural feature of a polypeptide which may serve to reverse thedirection of the backbone of a peptide or polypeptide. Where the loop isfound in a polypeptide and only alters the direction of the backbone, itmay comprise four or more amino acid residues. Oliva et al. haveidentified at least 5 classes of protein loops (J. Mol Biol 266 (4):814-830; 1997). Loops may be open or closed. Closed loops or “cyclic”loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsbetween the bridging moieties. Such bridging moieties may comprise acysteine-cysteine bridge (Cys-Cys) typical in polypeptides havingdisulfide bridges or alternatively bridging moieties may be non-proteinbased such as the dibromozylyl agents used herein.

As used herein when referring to polypeptides the term “half-loop”refers to a portion of an identified loop having at least half thenumber of amino acid resides as the loop from which it is derived. It isunderstood that loops may not always contain an even number of aminoacid residues. Therefore, in those cases where a loop contains or isidentified to comprise an odd number of amino acids, a half-loop of theodd-numbered loop will comprise the whole number portion or next wholenumber portion of the loop (number of amino acids of the loop/2+/−0.5amino acids). For example, a loop identified as a 7 amino acid loopcould produce half-loops of 3 amino acids or 4 amino acids(7/2=3.5+/−0.5 being 3 or 4).

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the term “half-domain”means a portion of an identified domain having at least half the numberof amino acid resides as the domain from which it is derived. It isunderstood that domains may not always contain an even number of aminoacid residues. Therefore, in those cases where a domain contains or isidentified to comprise an odd number of amino acids, a half-domain ofthe odd-numbered domain will comprise the whole number portion or nextwhole number portion of the domain (number of amino acids of thedomain/2+/−0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/−0.5 being 3 or 4). It is also understood thatsubdomains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids that compriseany of the domain types herein need not be contiguous along the backboneof the polypeptide (i.e., nonadjacent amino acids may fold structurallyto produce a domain, half-domain or subdomain).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a peptide or polypeptide that may be modified,manipulated, altered, derivatized or varied within the polypeptide basedmolecules of the present invention.

As used herein the terms “termini” or “terminus” when referring topolypeptides refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide to be encoded by the primary construct ormmRNA of the invention, any of several manipulations and/ormodifications of these features may be performed by moving, swapping,inverting, deleting, randomizing or duplicating. Furthermore, it isunderstood that manipulation of features may result in the same outcomeas a modification to the molecules of the invention. For example, amanipulation which involved deleting a domain would result in thealteration of the length of a molecule just as modification of a nucleicacid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis. Theresulting modified molecules may then be tested for activity using invitro or in vivo assays such as those described herein or any othersuitable screening assay known in the art.

According to the present invention, the polypeptides may comprise aconsensus sequence which is discovered through rounds ofexperimentation. As used herein a “consensus” sequence is a singlesequence which represents a collective population of sequences allowingfor variability at one or more sites.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest of this invention. Forexample, provided herein is any protein fragment (meaning an polypeptidesequence at least one amino acid residue shorter than a referencepolypeptide sequence but otherwise identical) of a reference protein 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids inlength. In another example, any protein that includes a stretch of about20, about 30, about 40, about 50, or about 100 amino acids which areabout 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 100% identical to any of the sequences described hereincan be utilized in accordance with the invention. In certainembodiments, a polypeptide to be utilized in accordance with theinvention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided or referenced herein.

Encoded Polypeptides of Interest

The primary constructs, modified nucleic acids or mmRNA of the presentinvention may be designed to encode polypeptides of interest such aspeptides and proteins.

In one embodiment, primary constructs, modified nucleic acids or mmRNAof the present invention may encode variant polypeptides which have acertain identity with a reference polypeptide sequence. As used herein,a “reference polypeptide sequence” refers to a starting polypeptidesequence. Reference sequences may be wild type sequences or any sequenceto which reference is made in the design of another sequence. A“reference polypeptide sequence” may, e.g., be any one of the proteinsequence listed in U.S. Provisional Patent Application No. 61/618,862,filed Apr. 2, 2012, entitled Modified Polynucleotides for the Productionof Biologics, U.S. Provisional Patent Application No. 61/681,645, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofBiologics, U.S. Provisional Patent Application No. 61/737,130, filedDec. 14, 2012, entitled Modified Polynucleotides for the Production ofBiologics, U.S. Provisional Patent Application No. 61/618,866, filedApr. 2, 2012, entitled Modified Polynucleotides for the Production ofAntibodies, U.S. Provisional Patent Application No. 61/681,647, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofAntibodies, U.S. Provisional Patent Application No. 61/737,134, filedDec. 14, 2012, entitled Modified Polynucleotides for the Production ofAntibodies, U.S. Provisional Patent Application No. 61/618,868, filedApr. 2, 2012, entitled Modified Polynucleotides for the Production ofVaccines, U.S. Provisional Patent Application No. 61/681,648, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofVaccines, U.S. Provisional Patent Application No. 61/737,135, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofVaccines, U.S. Provisional Patent Application No. 61/618,870, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofTherapeutic Proteins and Peptides, U.S. Provisional Patent ApplicationNo. 61/681,649, filed Aug. 10, 2012, entitled Modified Polynucleotidesfor the Production of Therapeutic Proteins and Peptides, U.S.Provisional Patent Application No. 61/737,139, filed Dec. 14, 2012,Modified Polynucleotides for the Production of Therapeutic Proteins andPeptides, U.S. Provisional Patent Application No. 61/618,873, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins, U.S. Provisional Patent Application No. 61/681,650,filed Aug. 10, 2012, entitled Modified Polynucleotides for theProduction of Secreted Proteins, U.S. Provisional Patent Application No.61/737,147, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Secreted Proteins, U.S. Provisional Patent ApplicationNo. 61/618,878, filed Apr. 2, 2012, entitled Modified Polynucleotidesfor the Production of Plasma Membrane Proteins, U.S. Provisional PatentApplication No. 61/681,654, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Plasma Membrane Proteins, U.S.Provisional Patent Application No. 61/737,152, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins, U.S. Provisional Patent Application No. 61/618,885, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/681,658, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins, U.S. Provisional Patent Application No. 61/737,155, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/618,896, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins, U.S. Provisional Patent Application No. 61/668,157, filed Jul.5, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/681,661, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins, U.S. Provisional Patent Application No. 61/737,160, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/618,911, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Nuclear Proteins, U.S. ProvisionalPatent Application No. 61/681,667, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Nuclear Proteins, U.S.Provisional Patent Application No. 61/737,168, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins, U.S. Provisional Patent Application No. 61/618,922, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/681,675, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/737,174, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/618,935, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/681,687, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/737,184, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/618,945, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/681,696, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/737,191, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Patent Application No. 61/618,953, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of ProteinsAssociated with Human Disease, U.S. Patent Application No. 61/681,704,filed Aug. 10, 2012, entitled Modified Polynucleotides for theProduction of Proteins Associated with Human Disease, U.S. PatentApplication No. 61/737,203, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, International Application No PCT/US2013/030062, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of Biologicsand Proteins Associated with Human Disease; International Application NoPCT/US2013/030063, filed Mar. 9, 2013, entitled ModifiedPolynucleotides; International Application No. PCT/US2013/030064,entitled Modified Polynucleotides for the Production of SecretedProteins; International Application No PCT/US2013/030059, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of MembraneProteins; International Application No. PCT/US2013/030066, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins; International Application No.PCT/US2013/030067, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Nuclear Proteins; International Application No.PCT/US2013/030060, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Proteins; International Application No.PCT/US2013/030061, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Proteins Associated with Human Disease;International Application No. PCT/US2013/030068, filed Mar. 9, 2013,entitled Modified Polynucleotides for the Production of CosmeticProteins and Peptides; International Application No. PCT/US2013/030070,filed Mar. 9, 2013, entitled Modified Polynucleotides for the Productionof Oncology-Related Proteins and Peptides; and International ApplicationNo. PCT/US2013/031821, filed Mar. 15, 2013, entitled In Vivo Productionof Proteins, the contents of each of which are herein incorporated byreference in their entireties.

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more peptides, as determined bycomparing the sequences. In the art, identity also means the degree ofsequence relatedness between peptides, as determined by the number ofmatches between strings of two or more amino acid residues. Identitymeasures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated peptides can be readily calculated by known methods. Suchmethods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

In some embodiments, the polypeptide variant may have the same or asimilar activity as the reference polypeptide. Alternatively, thevariant may have an altered activity (e.g., increased or decreased)relative to a reference polypeptide. Generally, variants of a particularpolynucleotide or polypeptide of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity tothat particular reference polynucleotide or polypeptide as determined bysequence alignment programs and parameters described herein and known tothose skilled in the art. Such tools for alignment include those of theBLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A.Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman(1997), “Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs”, Nucleic Acids Res. 25:3389-3402.) Other toolsare described herein, specifically in the definition of “identity.”

Default parameters in the BLAST algorithm include, for example, anexpect threshold of 10, Word size of 28, Match/Mismatch Scores 1, -2,Gap costs Linear. Any filter can be applied as well as a selection forspecies specific repeats, e.g., Homo sapiens.

In one embodiment, the polynucleotides, primary constructs, modifiednucleic acids and/or mmRNA may be used to treat a disease, disorderand/or condition in a subject.

In one embodiment, the polynucleotides, primary constructs, modifiednucleic acids and/or mmRNA may be used to reduce, eliminate or preventtumor growth in a subject.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be used to reduce and/or ameliorate at least one symptom of cancerin a subject. A symptom of cancer may include, but is not limited to,weakness, aches and pains, fever, fatigue, weight loss, blood clots,increased blood calcium levels, low white blood cell count, short ofbreath, dizziness, headaches, hyperpigmentation, jaundice, erthema,pruritis, excessive hair growth, change in bowel habits, change inbladder function, long-lasting sores, white patches inside the mouth,white spots on the tongue, unusual bleeding or discharge, thickening orlump on parts of the body, indigestion, trouble swallowing, changes inwarts or moles, change in new skin and nagging cough or hoarseness.Further, the polynucleotides, primary constructs, modified nucleic acidand/or mmRNA may reduce a side-effect associated with cancer such as,but not limited to, chemo brain, peripheral neuropathy, fatigue,depression, nausea, vomiting, pain, anemia, lymphedema, infections,sexual side effects, reduced fertility or infertility, ostomics,insomnia and hair loss.

Terminal Architecture Modifications: Untranslated Regions (UTRs)

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon;whereas, the 3′UTR starts immediately following the stop codon andcontinues until the transcriptional termination signal. There is growingbody of evidence about the regulatory roles played by the UTRs in termsof stability of the nucleic acid molecule and translation. Theregulatory features of a UTR can be incorporated into the nucleic acidsor modified RNA of the present invention to enhance the stability of themolecule. The specific features can also be incorporated to ensurecontrolled down-regulation of the transcript in case they aremisdirected to undesired organs sites. The untranslated regions may beincorporated into a vector system which can produce mRNA and/or bedelivered to a cell, tissue and/or organism to produce a polypeptide ofinterest.

5′ UTR and Translation Initiation

Natural 5′UTRs bear features which play roles in for translationinitiation. They harbor signatures like Kozak sequences which arecommonly known to be involved in the process by which the ribosomeinitiates translation of many genes. Kozak sequences have the consensusCCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three basesupstream of the start codon (AUG), which is followed by another ‘G’.5′UTR also have been known to form secondary structures which areinvolved in elongation factor binding.

5′UTR secondary structures involved in elongation factor binding caninteract with other RNA binding molecules in the 5′UTR or 3′UTR toregulate gene expression. For example, the elongation factor EIF4A2binding to a secondarily structured element in the 5′UTR is necessaryfor microRNA mediated repression (Meijer H A et al., Science, 2013, 340,82-85, herein incorporated by reference in its entirety). The differentsecondary structures in the 5′UTR can be incorporated into the flankingregion to either stabilize or selectively destalized mRNAs in specifictissues or cells.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of the nucleic acids or mRNA of the invention. Forexample, introduction of 5′ UTR of liver-expressed mRNA, such asalbumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alphafetoprotein, erythropoietin, or Factor VIII, could be used to enhanceexpression of a nucleic acid molecule, such as a mmRNA, in hepatic celllines or liver. Likewise, use of 5′ UTR from other tissue-specific mRNAto improve expression in that tissue is possible—for muscle (MyoD,Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1,CD36), for myeloid cells (C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1,i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4,ACRP30, adiponectin) and for lung epithelial cells (SP-A/B/C/D).

Other non-UTR sequences may be incorporated into the 5′ (or 3′ UTR)UTRs. For example, introns or portions of introns sequences may beincorporated into the flanking regions of the nucleic acids or mRNA ofthe invention. Incorporation of intronic sequences may increase proteinproduction as well as mRNA levels.

In one embodiment, at least one fragment of IRES sequences from a GTXgene may be included in the 5′UTR. As a non-limiting example, thefragment may be an 18 nucleotide sequence from the IRES of the GTX gene.As another non-limiting example, an 18 nucleotide sequence fragment fromthe IRES sequence of a GTX gene may be tandemly repeated in the 5′UTR ofa polynucleotide described herein. The 18 nucleotide sequence may berepeated in the 5′UTR at least one, at least twice, at least threetimes, at least four times, at least five times, at least six times, atleast seven times, at least eight times, at least nine times or morethan ten times

In one embodiment, a 5′UTR may include at least five 18 nucleotidefragments of IRES sequences from a GTX gene may be included in the 5′UTR(see e.g., the 18 nucleotide fragment described in Table 62).

Nucleotides may be mutated, replaced and/or removed from the 5′ (or 3′)UTRs. For example, one or more nucleotides upstream of the start codonmay be replaced with another nucleotide. The nucleotide or nucleotidesto be replaced may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 ormore than 60 nucleotides upstream of the start codon. As anotherexample, one or more nucleotides upstream of the start codon may beremoved from the UTR.

In one embodiment, at least one purine upstream of the start codon maybe replaced with a pyrimidine. The purine to be replaced may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 55, 60 or more than 60 nucleotidesupstream of the start codon. As a non-limiting example, an adenine whichis three nucleotides upstream of the start codon may be replaced with athymine. As another non-limiting example, an adenine which is ninenucleotides upstream of the start codon may be replaced with a thymine.

In one embodiment, at least one nucleotide upstream of the start codonmay be removed from the UTR. In one aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,40, 45, 50, 55, 60 or more than 60 nucleotides upstream of the startcodon may be removed from the UTR of the polynucleotides describedherein. As a non-limiting example, the nine nucleotides upstream of thestart codon may be removed from the UTR (See e.g., the G-CSF 9de15′construct described in Table 60).

5′UTR, 3′UTR and Translation Enhancer Elements (TEEs)

In one embodiment, the 5′UTR of the polynucleotides, primary constructs,modified nucleic acids and/or mmRNA may include at least onetranslational enhancer polynucleotide, translation enhancer element,translational enhancer elements (collectively referred to as “TEE”s). Asa non-limiting example, the TEE may be located between the transcriptionpromoter and the start codon. The polynucleotides, primary constructs,modified nucleic acids and/or mmRNA with at least one TEE in the 5′UTRmay include a cap at the 5′UTR. Further, at least one TEE may be locatedin the 5′UTR of polynucleotides, primary constructs, modified nucleicacids and/or mmRNA undergoing cap-dependent or cap-independenttranslation.

The term “translational enhancer element” or “translation enhancerelement” (herein collectively referred to as “TEE”) refers to sequencesthat increase the amount of polypeptide or protein produced from anmRNA.

In one aspect, TEEs are conserved elements in the UTR which can promotetranslational activity of a nucleic acid such as, but not limited to,cap-dependent or cap-independent translation. The conservation of thesesequences has been previously shown by Panek et al (Nucleic AcidsResearch, 2013, 1-10; herein incorporated by reference in its entirety)across 14 species including humans.

In one embodiment, the TEE may be any of the TEEs listed in Table 32 inExample 45, including portion and/or fragments thereof. The TEE sequencemay include at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99% or more than 99% of the TEE sequences disclosed in Table 32and/or the TEE sequence may include a 5-30 nucleotide fragment, a 5-25nucleotide fragment, a 5-20 nucleotide fragment, a 5-15 nucleotidefragment, a 5-10 nucleotide fragment of the TEE sequences disclosed inTable 32.

In one non-limiting example, the TEEs known may be in the 5′-leader ofthe Gtx homeodomain protein (Chappell et al., Proc. Natl. Acad. Sci. USA101:9590-9594, 2004, herein incorporated by reference in theirentirety).

In another non-limiting example, TEEs are disclosed as SEQ ID NOs: 1-35in US Patent Publication No. US20090226470, SEQ ID NOs: 1-35 in USPatent Publication US20130177581, SEQ ID NOs: 1-35 in InternationalPatent Publication No. WO2009075886, SEQ ID NOs: 1-5, and 7-645 inInternational Patent Publication No. WO2012009644, SEQ ID NO: 1 inInternational Patent Publication No. WO1999024595, SEQ ID NO: 1 in U.S.Pat. No. 6,310,197, and SEQ ID NO: 1 in U.S. Pat. No. 6,849,405, each ofwhich is herein incorporated by reference in its entirety.

In yet another non-limiting example, the TEE may be an internal ribosomeentry site (IRES), HCV-IRES or an IRES element such as, but not limitedto, those described in U.S. Pat. No. 7,468,275, US Patent PublicationNos. US20070048776 and US20110124100 and International PatentPublication Nos. WO2007025008 and WO2001055369, each of which is hereinincorporated by reference in its entirety. The IRES elements mayinclude, but are not limited to, the Gtx sequences (e.g., Gtx9-nt,Gtx8-nt, Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci.USA 101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278, 2005) andin US Patent Publication Nos. US20070048776 and US20110124100 andInternational Patent Publication No. WO2007025008, each of which isherein incorporated by reference in its entirety.

“Translational enhancer polynucleotides” or “translation enhancerpolynucleotide sequences” are polynucleotides which include one or moreof the specific TEE exemplified herein and/or disclosed in the art (seee.g., U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No.7,456,273, U.S. Pat. No. 7,183,395, US20090226470, US20070048776,US20110124100, US20090093049, US20130177581, WO2009075886, WO2007025008,WO2012009644, WO2001055371 WO1999024595, and EP2610341A1 andEP2610340A1; each of which is herein incorporated by reference in itsentirety) or their variants, homologs or functional derivatives. One ormultiple copies of a specific TEE can be present in the polynucleotides,primary constructs, modified nucleic acids and/or mmRNA. The TEEs in thetranslational enhancer polynucleotides can be organized in one or moresequence segments. A sequence segment can harbor one or more of thespecific TEEs exemplified herein, with each TEE being present in one ormore copies. When multiple sequence segments are present in atranslational enhancer polynucleotide, they can be homogenous orheterogeneous. Thus, the multiple sequence segments in a translationalenhancer polynucleotide can harbor identical or different types of thespecific TEEs exemplified herein, identical or different number ofcopies of each of the specific TEEs, and/or identical or differentorganization of the TEEs within each sequence segment.

In one embodiment, the polynucleotides, primary constructs, modifiednucleic acids and/or mmRNA may include at least one TEE that isdescribed in International Patent Publication No. WO1999024595,WO2012009644, WO2009075886, WO2007025008, WO1999024595, European PatentPublication No. EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197,U.S. Pat. No. 6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No.7,183,395, US Patent Publication No. US20090226470, US20110124100,US20070048776, US20090093049, and US20130177581 each of which is hereinincorporated by reference in its entirety. The TEE may be located in the5′UTR of the polynucleotides, primary constructs, modified nucleic acidsand/or mmRNA.

In another embodiment, the polynucleotides, primary constructs, modifiednucleic acids and/or mmRNA may include at least one TEE that has atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95% or atleast 99% identity with the TEEs described in US Patent Publication Nos.US20090226470, US20070048776, US20130177581 and US20110124100,International Patent Publication No. WO1999024595, WO2012009644,WO2009075886 and WO2007025008, European Patent Publication No.EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No.6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395, each ofwhich is herein incorporated by reference in its entirety.

In one embodiment, the 5′UTR of the polynucleotides, primary constructs,modified nucleic acids and/or mmRNA may include at least 1, at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18 at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, at least 30, at least 35, at least 40, at least 45, at least 50, atleast 55 or more than 60 TEE sequences. The TEE sequences in the 5′UTRof the polynucleotides, primary constructs, modified nucleic acidsand/or mmRNA of the present invention may be the same or different TEEsequences. The TEE sequences may be in a pattern such as ABABAB orAABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, ormore than three times. In these patterns, each letter, A, B, or Crepresent a different TEE sequence at the nucleotide level.

In one embodiment, the 5′UTR may include a spacer to separate two TEEsequences. As a non-limiting example, the spacer may be a 15 nucleotidespacer and/or other spacers known in the art. As another non-limitingexample, the 5′UTR may include a TEE sequence-spacer module repeated atleast once, at least twice, at least 3 times, at least 4 times, at least5 times, at least 6 times, at least 7 times, at least 8 times and atleast 9 times or more than 9 times in the 5′UTR.

In another embodiment, the spacer separating two TEE sequences mayinclude other sequences known in the art which may regulate thetranslation of the polynucleotides, primary constructs, modified nucleicacids and/or mmRNA of the present invention such as, but not limited to,miR sequences described herein (e.g., miR binding sites and miR seeds).As a non-limiting example, each spacer used to separate two TEEsequences may include a different miR sequence or component of a miRsequence (e.g., miR seed sequence).

In one embodiment, the TEE in the 5′UTR of the polynucleotides, primaryconstructs, modified nucleic acids and/or mmRNA of the present inventionmay include at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99% or more than 99% of the TEE sequences disclosed in US PatentPublication Nos. US20090226470, US20070048776, US20130177581 andUS20110124100, International Patent Publication No. WO1999024595,WO2012009644, WO2009075886 and WO2007025008, European Patent PublicationNo. EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No.6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395. In anotherembodiment, the TEE in the 5′UTR of the polynucleotides, primaryconstructs, modified nucleic acids and/or mmRNA of the present inventionmay include a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotidefragment of the TEE sequences disclosed in US Patent Publication Nos.US20090226470, US20070048776, US20130177581 and US20110124100,International Patent Publication No. WO1999024595, WO2012009644,WO2009075886 and WO2007025008, European Patent Publication No.EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No.6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395; each ofwhich are herein incorporated by reference in their entirety.

In one embodiment, the TEE in the 5′UTR of the polynucleotides, primaryconstructs, modified nucleic acids and/or mmRNA of the present inventionmay include at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99% or more than 99% of the TEE sequences disclosed in Chappell etal. (Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004) and Zhou et al.(PNAS 102:6273-6278, 2005), in Supplemental Table 1 and in SupplementalTable 2 disclosed by Wellensiek et al (Genome-wide profiling of humancap-independent translation-enhancing elements, Nature Methods, 2013;DOI:10.1038/NMETH.2522); each of which is herein incorporated byreference in its entirety. In another embodiment, the TEE in the 5′UTRof the polynucleotides, primary constructs, modified nucleic acidsand/or mmRNA of the present invention may include a 5-30 nucleotidefragment, a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequencesdisclosed in Chappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594,2004) and Zhou et al. (PNAS 102:6273-6278, 2005), in Supplemental Table1 and in Supplemental Table 2 disclosed by Wellensiek et al (Genome-wideprofiling of human cap-independent translation-enhancing elements,Nature Methods, 2013; DOI:10.1038/NMETH.2522); each of which is hereinincorporated by reference in its entirety.

In one embodiment, the TEE used in the 5′UTR of the polynucleotides,primary constructs, modified nucleic acids and/or mmRNA of the presentinvention is an IRES sequence such as, but not limited to, thosedescribed in U.S. Pat. No. 7,468,275 and International PatentPublication No. WO2001055369, each of which is herein incorporated byreference in its entirety.

In one embodiment, the TEEs used in the 5′UTR of the polynucleotides,primary constructs, modified nucleic acids and/or mmRNA of the presentinvention may be identified by the methods described in US PatentPublication No. US20070048776 and US20110124100 and International PatentPublication Nos. WO2007025008 and WO2012009644, each of which is hereinincorporated by reference in its entirety.

In another embodiment, the TEEs used in the 5′UTR of thepolynucleotides, primary constructs, modified nucleic acids and/or mmRNAof the present invention may be a transcription regulatory elementdescribed in U.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, USPatent Publication No. US20090093049, and International Publication No.WO2001055371, each of which is herein incorporated by reference in theirentirety. The transcription regulatory elements may be identified bymethods known in the art, such as, but not limited to, the methodsdescribed in U.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, USPatent Publication No. US20090093049, and International Publication No.WO2001055371, each of which is herein incorporated by reference in theirentirety.

In yet another embodiment, the TEE used in the 5′UTR of thepolynucleotides, primary constructs, modified nucleic acids and/or mmRNAof the present invention is an oligonucleotide or portion thereof asdescribed in U.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, USPatent Publication No. US20090093049, and International Publication No.WO2001055371, each of which is herein incorporated by reference in theirentirety.

The 5′ UTR comprising at least one TEE described herein may beincorporated in a monocistronic sequence such as, but not limited to, avector system or a nucleic acid vector. As a non-limiting example, thevector systems and nucleic acid vectors may include those described inU.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, US PatentPublication No. US20070048776, US20090093049 and US20110124100 andInternational Patent Publication Nos. WO2007025008 and WO2001055371,each of which is herein incorporated by reference in its entirety.

In one embodiment, the TEEs described herein may be located in the 5′UTRand/or the 3′UTR of the polynucleotides, primary constructs, modifiednucleic acids and/or mmRNA. The TEEs located in the 3′UTR may be thesame and/or different than the TEEs located in and/or described forincorporation in the 5′UTR.

In one embodiment, the 3′UTR of the polynucleotides, primary constructs,modified nucleic acids and/or mmRNA may include at least 1, at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18 at least 19, atleast 20, at least 21, at least 22, at least 23, at least 24, at least25, at least 30, at least 35, at least 40, at least 45, at least 50, atleast 55 or more than 60 TEE sequences. The TEE sequences in the 3′UTRof the polynucleotides, primary constructs, modified nucleic acidsand/or mmRNA of the present invention may be the same or different TEEsequences. The TEE sequences may be in a pattern such as ABABAB orAABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, ormore than three times. In these patterns, each letter, A, B, or Crepresent a different TEE sequence at the nucleotide level.

In one embodiment, the 3′UTR may include a spacer to separate two TEEsequences. As a non-limiting example, the spacer may be a 15 nucleotidespacer and/or other spacers known in the art. As another non-limitingexample, the 3′UTR may include a TEE sequence-spacer module repeated atleast once, at least twice, at least 3 times, at least 4 times, at least5 times, at least 6 times, at least 7 times, at least 8 times and atleast 9 times or more than 9 times in the 3′UTR.

In another embodiment, the spacer separating two TEE sequences mayinclude other sequences known in the art which may regulate thetranslation of the polynucleotides, primary constructs, modified nucleicacids and/or mmRNA of the present invention such as, but not limited to,miR sequences described herein (e.g., miR binding sites and miR seeds).As a non-limiting example, each spacer used to separate two TEEsequences may include a different miR sequence or component of a miRsequence (e.g., miR seed sequence).

In one embodiment, the incorporation of a miR sequence and/or a TEEsequence changes the shape of the stem loop region which may increaseand/or decrease translation. (see e.g, Kedde et al. A Pumilio-inducedRNA structure switch in p27-3′UTR controls miR-221 and miR-22accessibility. Nature Cell Biology. 2010, herein incorporated byreference in its entirety).

Heterologous 5′UTRs

A 5′ UTR may be provided as a flanking region to the modified nucleicacids (mRNA), enhanced modified RNA or ribonucleic acids of theinvention. 5′UTR may be homologous or heterologous to the coding regionfound in the modified nucleic acids (mRNA), enhanced modified RNA orribonucleic acids of the invention. Multiple 5′ UTRs may be included inthe flanking region and may be the same or of different sequences. Anyportion of the flanking regions, including none, may be codon optimizedand any may independently contain one or more different structural orchemical modifications, before and/or after codon optimization.

Shown in Lengthy Table 21 in U.S. Provisional Application No.61/775,509, filed Mar. 9, 2013, entitled Heterologous UntranslatedRegions for mRNA and in Lengthy Table 21 and in Table 22 in U.S.Provisional Application No. 61/829,372, filed May 31, 2013, entitledHeterologous Untranslated Regions for mRNA, the contents of each ofwhich is herein incorporated by reference in its entirety, is a listingof the start and stop site of the modified nucleic acids (mRNA),enhanced modified RNA or ribonucleic acids of the invention. In Table 21each 5′UTR (5′UTR-005 to 5′UTR 68511) is identified by its start andstop site relative to its native or wild type (homologous) transcript(ENST; the identifier used in the ENSEMBL database).

Additional 5′UTR which may be used with the modified nucleic acids,enhanced modified RNA or ribonucleic acids of the invention are shown inthe present disclosure in Table 6, Table 38 and Table 41.

To alter one or more properties of the polynucleotides, primaryconstructs or mmRNA of the invention, 5′UTRs which are heterologous tothe coding region of the modified nucleic acids (mRNA), enhancedmodified RNA or ribonucleic acids of the invention are engineered intocompounds of the invention. The modified nucleic acids (mRNA), enhancedmodified RNA or ribonucleic acids are then administered to cells, tissueor organisms and outcomes such as protein level, localization and/orhalf life are measured to evaluate the beneficial effects theheterologous 5′UTR may have on the modified nucleic acids (mRNA),enhanced modified RNA or ribonucleic acids of the invention. Variants ofthe 5′ UTRs may be utilized wherein one or more nucleotides are added orremoved to the termini, including A, T, C or G. 5′UTRs may also becodon-optimized or modified in any manner described herein.

Incorporating microRNA Binding Sites

In one embodiment modified nucleic acids (mRNA), enhanced modified RNAor ribonucleic acids of the invention would not only encode apolypeptide but also a sensor sequence. Sensor sequences include, forexample, microRNA binding sites, transcription factor binding sites,structured mRNA sequences and/or motifs, artificial binding sitesengineered to act as pseudo-receptors for endogenous nucleic acidbinding molecules. Non-limiting examples, of polynucleotides comprisingat least one sensor sequence are described in co-pending and co-ownedU.S. Provisional Patent Application No. 61/753,661, filed Jan. 17, 2013,entitled Signal-Sensor Polynucleotide for the Alteration of CellularPhenotypes and Microenvironments, U.S. Provisional Patent ApplicationNo. 61/754,159, filed Jan. 18, 2013, entitled Signal-SensorPolynucleotide for the Alteration of Cellular Phenotypes andMicroenvironments, U.S. Provisional Patent Application No. 61/781,097,filed Mar. 14, 2013, entitled Signal-Sensor Polynucleotide for theAlteration of Cellular Phenotypes and Microenvironments, U.S.Provisional Patent Application No. 61/829,334, filed May 31, 2013,entitled Signal-Sensor Polynucleotide for the Alteration of CellularPhenotypes and Microenvironments, U.S. Provisional Patent ApplicationNo. 61/839,893, filed Jun. 27, 2013, entitled Signal-SensorPolynucleotide for the Alteration of Cellular Phenotypes andMicroenvironments, U.S. Provisional Patent Application No. 61/842,733,filed Jul. 3, 2013, entitled Signal-Sensor Polynucleotide for theAlteration of Cellular Phenotypes and Microenvironment, and U.S.Provisional Patent Application No. 61/857,304, filed Jul. 23, 2013,entitled Signal-Sensor Polynucleotide for the Alteration of CellularPhenotypes and Microenvironment, the contents of each of which areherein incorporated by reference in its entirety.

In one embodiment, microRNA (miRNA) profiling of the target cells ortissues is conducted to determine the presence or absence of miRNA inthe cells or tissues.

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′UTR of nucleic acid molecules and down-regulate gene expressioneither by reducing nucleic acid molecule stability or by inhibitingtranslation. The modified nucleic acids (mRNA), enhanced modified RNA orribonucleic acids of the invention may comprise one or more microRNAtarget sequences, microRNA sequences, or microRNA seeds. Such sequencesmay correspond to any known microRNA such as those taught in USPublication US2005/0261218 and US Publication US2005/0059005, thecontents of which are incorporated herein by reference in theirentirety. As a non-limiting embodiment, known microRNAs, their sequencesand seed sequences in human genome are listed below in Table 11.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked by an adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. The bases of themicroRNA seed have complete complementarity with the target sequence. Byengineering microRNA target sequences into the 3′UTR of nucleic acids ormRNA of the invention one can target the molecule for degradation orreduced translation, provided the microRNA in question is available.This process will reduce the hazard of off target effects upon nucleicacid molecule delivery. Identification of microRNA, microRNA targetregions, and their expression patterns and role in biology have beenreported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand andCheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia2012 26:404-413 (2011 Dec. 20. doi: 10.1038/leu.2011.356); Bartel Cell2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner andNaldini, Tissue Antigens. 2012 80:393-403 and all references therein;each of which is herein incorporated by reference in its entirety).

For example, if the mRNA is not intended to be delivered to the liverbut ends up there, then miR-122, a microRNA abundant in liver, caninhibit the expression of the gene of interest if one or multiple targetsites of miR-122 are engineered into the 3′UTR of the modified nucleicacids, enhanced modified RNA or ribonucleic acids. Introduction of oneor multiple binding sites for different microRNA can be engineered tofurther decrease the longevity, stability, and protein translation of amodified nucleic acids, enhanced modified RNA or ribonucleic acids. Asused herein, the term “microRNA site” refers to a microRNA target siteor a microRNA recognition site, or any nucleotide sequence to which amicroRNA binds or associates. It should be understood that “binding” mayfollow traditional Watson-Crick hybridization rules or may reflect anystable association of the microRNA with the target sequence at oradjacent to the microRNA site.

Conversely, for the purposes of the modified nucleic acids, enhancedmodified RNA or ribonucleic acids of the present invention, microRNAbinding sites can be engineered out of (i.e. removed from) sequences inwhich they naturally occur in order to increase protein expression inspecific tissues. For example, miR-122 binding sites may be removed toimprove protein expression in the liver.

In one embodiment, the modified nucleic acids, enhanced modified RNA orribonucleic acids of the present invention may include at least onemiRNA-binding site in the 3′UTR in order to direct cytotoxic orcytoprotective mRNA therapeutics to specific cells such as, but notlimited to, normal and/or cancerous cells (e.g., HEP3B or SNU449).

In another embodiment, the modified nucleic acids, enhanced modified RNAor ribonucleic acids of the present invention may include threemiRNA-binding sites in the 3′UTR in order to direct cytotoxic orcytoprotective mRNA therapeutics to specific cells such as, but notlimited to, normal and/or cancerous cells (e.g., HEP3B or SNU449).

Regulation of expression in multiple tissues can be accomplished throughintroduction or removal or one or several microRNA binding sites. Shownbelow in Table 12, microRNAs which are differentially expressed indifferent tissues and cells, and often associated with different typesof diseases (e.g. cancer cells). The decision of removal or insertion ofmicroRNA binding sites, or any combination, is dependent on microRNAexpression patterns and their profilings in diseases.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126).

Specifically, microRNAs are known to be differentially expressed inimmune cells (also called hematopoietic cells), such as antigenpresenting cells (APCs) (e.g. dendritic cells and macrophages),macrophages, monocytes, B lymphocytes, T lymphocytes, granuocytes,natural killer cells, etc. Immune cell specific microRNAs are involvedin immunogenicity, autoimmunity, the immune-response to infection,inflammation, as well as unwanted immune response after gene therapy andtissue/organ transplantation. Immune cells specific microRNAs alsoregulate many aspects of development, proliferation, differentiation andapoptosis of hematopoietic cells (immune cells). For example, miR-142and miR-146 are exclusively expressed in the immune cells, particularlyabundant in myeloid dendritic cells. It was demonstrated in the art thatthe immune response to exogenous nucleic acid molecules was shut-off byadding miR-142 binding sites to the 3′UTR of the delivered geneconstruct, enabling more stable gene transfer in tissues and cells.miR-142 efficiently degrades the exogenous mRNA in antigen presentingcells and suppresses cytotoxic elimination of transduced cells (Annoni Aet al., blood, 2009, 114, 5152-5161; Brown B D, et al., Nat med. 2006,12(5), 585-591; Brown B D, et al., blood, 2007, 110(13): 4144-4152, eachof which is herein incorporated by reference in its entirety).

An antigen-mediated immune response can refer to an immune responsetriggered by foreign antigens, which, when entering an organism, areprocessed by the antigen presenting cells and displayed on the surfaceof the antigen presenting cells. T cells can recognize the presentedantigen and induce a cytotoxic elimination of cells that express theantigen.

Introducing the miR-142 binding site into the 3′-UTR of a polypeptide ofthe present invention can selectively repress the gene expression in theantigen presenting cells through miR-142 mediated mRNA degradation,limiting antigen presentation in APCs (e.g. dendritic cells) and therebypreventing antigen-mediated immune response after the delivery of thepolynucleotides. The polynucleotides are therefore stably expressed intarget tissues or cells without triggering cytotoxic elimination.

In one embodiment, microRNAs binding sites that are known to beexpressed in immune cells, in particular, the antigen presenting cells,can be engineered into the polynucleotide to suppress the expression ofthe sensor-signal polynucleotide in APCs through microRNA mediated RNAdegradation, subduing the antigen-mediated immune response, while theexpression of the polynucleotide is maintained in non-immune cells wherethe immune cell specific microRNAs are not expressed. For example, toprevent the immunogenic reaction caused by a liver specific proteinexpression, the miR-122 binding site can be removed and the miR-142(and/or mirR-146) binding sites can be engineered into the 3-UTR of thepolynucleotide.

To further drive the selective degradation and suppression of mRNA inAPCs and macrophage, the polynucleotide may include another negativeregulatory element in the 3-UTR, either alone or in combination withmir-142 and/or mir-146 binding sites. As a non-limiting example, oneregulatory element is the Constitutive Decay Elements (CDEs).

Immune cells specific microRNAs include, but are not limited to,hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p,hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p,hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-1-3p,hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p, miR-125b-2-3p,miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p,miR-142-3p, miR-142-5p, miR-143-3p, miR-143-5p, miR-146a-3p,miR-146a-5p, miR-146b-3p, miR-146b-5p, miR-147a, miR-147b, miR-148a-5p,miR-148a-3p, miR-150-3p, miR-150-5p, miR-151b, miR-155-3p, miR-155-5p,miR-15a-3p, miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p,miR-16-2-3p, miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p,miR-181a-2-3p, miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p,miR-21-5p, miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p,miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p,miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p, miR-27b-5p,miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p, miR-29b-1-5p,miR-29b-2-5p, miR-29c-3p, miR-29c-5p, miR-30e-3p, miR-30e-5p,miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p, miR-345-5p, miR-346,miR-34a-3p, miR-34a-5p, miR-363-3p, miR-363-5p, miR-372, miR-377-3p,miR-377-5p, miR-493-3p, miR-493-5p, miR-542, miR-548b-5p, miR548c-5p,miR-548i, miR-548j, miR-548n, miR-574-3p, miR-598, miR-718, miR-935,miR-99a-3p, miR-99a-5p, miR-99b-3p and miR-99b-5p. microRNAs that areenriched in specific types of immune cells are listed in Table 13.Furthermore, novel microRNAs are discovered in the immune cells in theart through micro-array hybridization and microtome analysis (Jima D Det al, Blood, 2010, 116:e118-e127; Vaz C et al., BMC Genomics, 2010,11,288, the content of each of which is incorporated herein by referencein its entirety.)

MicroRNAs that are known to be expressed in the liver include, but arenot limited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p,miR-1228-5p, miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p,miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p,miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, miR-939-5p.MicroRNA binding sites from any liver specific microRNA can beintroduced to or removed from the polynucleotides to regulate theexpression of the polynucleotides in the liver. Liver specific microRNAsbinding sites can be engineered alone or further in combination withimmune cells (e.g. APCs) microRNA binding sites in order to preventimmune reaction against protein expression in the liver.

MicroRNAs that are known to be expressed in the lung include, but arenot limited to, let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p,miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p,miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p,miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p,miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p,miR-381-5p. MicroRNA binding sites from any lung specific microRNA canbe introduced to or removed from the polynucleotide to regulate theexpression of the polynucleotide in the lung. Lung specific microRNAsbinding sites can be engineered alone or further in combination withimmune cells (e.g. APCs) microRNA binding sites in order to prevent animmune reaction against protein expression in the lung.

MicroRNAs that are known to be expressed in the heart include, but arenot limited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p,miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p,miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p,miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p and miR-92b-5p. MicroRNAbinding sites from any heart specific microRNA can be introduced to orremoved from the polynucleotides to regulate the expression of thepolynucleotides in the heart. Heart specific microRNAs binding sites canbe engineered alone or further in combination with immune cells (e.g.APCs) microRNA binding sites to prevent an immune reaction againstprotein expression in the heart.

MicroRNAs that are known to be expressed in the nervous system include,but are not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p,miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p,miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p,miR-137, miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153,miR-181c-3p, miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b,miR-212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p,miR-23a-5p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p,miR-30c-2-3p, miR-30c-5p, miR-30d-3p, miR-30d-5p, miR-329, miR-342-3p,miR-3665, miR-3666, miR-380-3p, miR-380-5p, miR-383, miR-410,miR-425-3p, miR-425-5p, miR-454-3p, miR-454-5p, miR-483, miR-510,miR-516a-3p, miR-548b-5p, miR-548c-5p, miR-571, miR-7-1-3p, miR-7-2-3p,miR-7-5p, miR-802, miR-922, miR-9-3p and miR-9-5p. MicroRNAs enriched inthe nervous system further include those specifically expressed inneurons, including, but not limited to, miR-132-3p, miR-132-3p,miR-148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p,miR-212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p,miR-325, miR-326, miR-328, miR-922 and those specifically expressed inglial cells, including, but not limited to, miR-1250, miR-219-1-3p,miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p,miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, miR-657.MicroRNA binding sites from any CNS specific microRNA can be introducedto or removed from the polynucleotides to regulate the expression of thepolynucleotide in the nervous system. Nervous system specific microRNAsbinding sites can be engineered alone or further in combination withimmune cells (e.g. APCs) microRNA binding sites in order to preventimmune reaction against protein expression in the nervous system.

MicroRNAs that are known to be expressed in the pancreas include, butare not limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p,miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p,miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375,miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p and miR-944. MicroRNAbinding sites from any pancreas specific microRNA can be introduced toor removed from the polynucleotide to regulate the expression of thepolynucleotide in the pancreas. Pancreas specific microRNAs bindingsites can be engineered alone or further in combination with immunecells (e.g. APCs) microRNA binding sites in order to prevent an immunereaction against protein expression in the pancreas.

MicroRNAs that are known to be expressed in the kidney further include,but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p,miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p,miR-204-5p, miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p,miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p,miR30c-5p, miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5pand miR-562. MicroRNA binding sites from any kidney specific microRNAcan be introduced to or removed from the polynucleotide to regulate theexpression of the polynucleotide in the kidney. Kidney specificmicroRNAs binding sites can be engineered alone or further incombination with immune cells (e.g. APCs) microRNA binding sites toprevent an immune reaction against protein expression in the kidney.

MicroRNAs that are known to be expressed in the muscle further include,but are not limited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a,miR-133b, miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p,miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p andmiR-25-5p. MicroRNA binding sites from any muscle specific microRNA canbe introduced to or removed from the polynucleotide to regulate theexpression of the polynucleotide in the muscle. Muscle specificmicroRNAs binding sites can be engineered alone or further incombination with immune cells (e.g. APCs) microRNA binding sites toprevent an immune reaction against protein expression in the muscle.

MicroRNAs are differentially expressed in different types of cells, suchas endothelial cells, epithelial cells and adipocytes. For example,microRNAs that are expressed in endothelial cells include, but are notlimited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p,miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p,miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17-3p, miR-18a-3p, miR-18a-5p,miR-19a-3p, miR-19a-5p, miR-19b-1-5p, miR-19b-2-5p, miR-19b-3p,miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p, miR-21-5p,miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p,miR-296-5p, miR-361-3p, miR-361-5p, miR-421, miR-424-3p, miR-424-5p,miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p, miR-92b-3p andmiR-92b-5p. Many novel microRNAs are discovered in endothelial cellsfrom deep-sequencing analysis (Voellenkle C et al., RNA, 2012, 18,472-484, herein incorporated by reference in its entirety) microRNAbinding sites from any endothelial cell specific microRNA can beintroduced to or removed from the polynucleotide to modulate theexpression of the polynucleotide in the endothelial cells in variousconditions.

For further example, microRNAs that are expressed in epithelial cellsinclude, but are not limited to, let-7b-3p, let-7b-5p, miR-1246,miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p,miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5pspecific in respiratory ciliated epithelial cells; let-7 family,miR-133a, miR-133b, miR-126 specific in lung epithelial cells;miR-382-3p, miR-382-5p specific in renal epithelial cells and miR-762specific in corneal epithelial cells. MicroRNA binding sites from anyepithelial cell specific MicroRNA can be introduced to or removed fromthe polynucleotide to modulate the expression of the polynucleotide inthe epithelial cells in various conditions.

In addition, a large group of microRNAs are enriched in embryonic stemcells, controlling stem cell self-renewal as well as the developmentand/or differentiation of various cell lineages, such as neural cells,cardiac, hematopoietic cells, skin cells, osteogenic cells and musclecells (Kuppusamy K T et al., Curr. Mol Med, 2013, 13(5), 757-764;Vidigal J A and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436;Goff L A et al., PLoS One, 2009, 4:e7192; Morin R D et al., Genome Res,2008, 18, 610-621; Yoo J K et al., Stem Cells Dev. 2012, 21(11),2049-2057, each of which is herein incorporated by reference in itsentirety). MicroRNAs abundant in embryonic stem cells include, but arenot limited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p,miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246,miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p,miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p,miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p,miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367-3p, miR-367-5p,miR-369-3p, miR-369-5p, miR-370, miR-371, miR-373, miR-380-5p,miR-423-3p, miR-423-5p, miR-486-5p, miR-520c-3p, miR-548e, miR-548f,miR-548g-3p, miR-548g-5p, miR-548i, miR-548k, miR-548l, miR-548m,miR-548n, miR-548o-3p, miR-548o-5p, miR-548p, miR-664a-3p, miR-664a-5p,miR-664b-3p, miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p,miR-885-5p, miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p,miR-99b-3p and miR-99b-5p. Many predicted novel microRNAs are discoveredby deep sequencing in human embryonic stem cells (Morin R D et al.,Genome Res, 2008, 18, 610-621; Goff L A et al., PLoS One, 2009, 4:e7192;Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each ofwhich is incorporated herein by references in its entirety).

In one embodiment, the binding sites of embryonic stem cell specificmicroRNAs can be included in or removed from the 3-UTR of thepolynucleotide to modulate the development and/or differentiation ofembryonic stem cells, to inhibit the senescence of stem cells in adegenerative condition (e.g. degenerative diseases), or to stimulate thesenescence and apoptosis of stem cells in a disease condition (e.g.cancer stem cells).

Many microRNA expression studies are conducted in the art to profile thedifferential expression of microRNAs in various cancer cells/tissues andother diseases. Some microRNAs are abnormally over-expressed in certaincancer cells and others are under-expressed. For example, microRNAs aredifferentially expressed in cancer cells (WO2008/154098, US2013/0059015,US2013/0042333, WO2011/157294); cancer stem cells (US2012/0053224);pancreatic cancers and diseases (US2009/0131348, US2011/0171646,US2010/0286232, U.S. Pat. No. 8,389,210); asthma and inflammation (U.S.Pat. No. 8,415,096); prostate cancer (US2013/0053264); hepatocellularcarcinoma (WO2012/151212, US2012/0329672, WO2008/054828, U.S. Pat. No.8,252,538); lung cancer cells (WO2011/076143, WO2013/033640,WO2009/070653, US2010/0323357); cutaneous T cell lymphoma(WO2013/011378); colorectal cancer cells (WO2011/0281756,WO2011/076142); cancer positive lympho nodes (WO2009/100430,US2009/0263803); nasopharyngeal carcinoma (EP2112235); chronicobstructive pulmonary disease (US2012/0264626, US2013/0053263); thyroidcancer (WO2013/066678); ovarian cancer cells (US2012/0309645,WO2011/095623); breast cancer cells (WO2008/154098, WO2007/081740,US2012/0214699), leukemia and lymphoma (WO2008/073915, US2009/0092974,US2012/0316081, US2012/0283310, WO2010/018563, the content of each ofwhich is incorporated herein by reference in their entirety.)

As a non-limiting example, microRNA sites that are over-expressed incertain cancer and/or tumor cells can be removed from the 3-UTR of thepolynucleotide encoding the polypeptide of interest, restoring theexpression suppressed by the over-expressed microRNAs in cancer cells,thus ameliorating the corresponsive biological function, for instance,transcription stimulation and/or repression, cell cycle arrest,apoptosis and cell death. Normal cells and tissues, wherein microRNAsexpression is not up-regulated, will remain unaffected.

MicroRNA can also regulate complex biological processes such asangiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 201118:171-176). In the modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention, binding sites for microRNAs that areinvolved in such processes may be removed or introduced, in order totailor the expression of the modified nucleic acids, enhanced modifiedRNA or ribonucleic acids expression to biologically relevant cell typesor to the context of relevant biological processes. In this context, themRNA are defined as auxotrophic mRNA.

MicroRNA gene regulation may be influenced by the sequence surroundingthe microRNA such as, but not limited to, the species of the surroundingsequence, the type of sequence (e.g., heterologous, homologous andartificial), regulatory elements in the surrounding sequence and/orstructural elements in the surrounding sequence. The microRNA may beinfluenced by the 5′UTR and/or the 3′UTR. As a non-limiting example, anon-human 3′UTR may increase the regulatory effect of the microRNAsequence on the expression of a polypeptide of interest compared to ahuman 3′UTR of the same sequence type.

In one embodiment, other regulatory elements and/or structural elementsof the 5′-UTR can influence microRNA mediated gene regulation. Oneexample of a regulatory element and/or structural element is astructured IRES (Internal Ribosome Entry Site) in the 5′UTR, which isnecessary for the binding of translational elongation factors toinitiate protein translation. EIF4A2 binding to this secondarilystructured element in the 5′UTR is necessary for microRNA mediated geneexpression (Meijer H A et al., Science, 2013, 340, 82-85, hereinincorporated by reference in its entirety). The modified nucleic acids,enhanced modified RNA or ribonucleic acids of the invention can furtherbe modified to include this structured 5′-UTR in order to enhancemicroRNA mediated gene regulation.

At least one microRNA site can be engineered into the 3′ UTR of themodified nucleic acids, enhanced modified RNA or ribonucleic acids ofthe present invention. In this context, at least two, at least three, atleast four, at least five, at least six, at least seven, at least eight,at least nine, at least ten or more microRNA sites may be engineeredinto the 3′ UTR of the ribonucleic acids of the present invention. Inone embodiment, the microRNA sites incorporated into the modifiednucleic acids, enhanced modified RNA or ribonucleic acids may be thesame or may be different microRNA sites. In another embodiment, themicroRNA sites incorporated into the modified nucleic acids, enhancedmodified RNA or ribonucleic acids may target the same or differenttissues in the body. As a non-limiting example, through the introductionof tissue-, cell-type-, or disease-specific microRNA binding sites inthe 3′ UTR of a modified nucleic acid mRNA, the degree of expression inspecific cell types (e.g. hepatocytes, myeloid cells, endothelial cells,cancer cells, etc.) can be reduced.

In one embodiment, a microRNA site can be engineered near the 5′terminus of the 3′UTR, about halfway between the 5′ terminus and 3′terminus of the 3′UTR and/or near the 3′ terminus of the 3′UTR. As anon-limiting example, a microRNA site may be engineered near the 5′terminus of the 3′UTR and about halfway between the 5′ terminus and 3′terminus of the 3′UTR. As another non-limiting example, a microRNA sitemay be engineered near the 3′ terminus of the 3′UTR and about halfwaybetween the 5′ terminus and 3′ terminus of the 3′UTR. As yet anothernon-limiting example, a microRNA site may be engineered near the 5′terminus of the 3′UTR and near the 3′ terminus of the 3′UTR.

In another embodiment, a 3′UTR can comprise 4 microRNA sites. ThemicroRNA sites may be complete microRNA binding sites, microRNA seedsequences and/or microRNA binding site sequences without the seedsequence.

In one embodiment, a nucleic acid of the invention may be engineered toinclude at least one microRNA in order to dampen the antigenpresentation by antigen presenting cells. The microRNA may be thecomplete microRNA sequence, the microRNA seed sequence, the microRNAsequence without the seed or a combination thereof. As a non-limitingexample, the microRNA incorporated into the nucleic acid may be specificto the hematopoietic system. As another non-limiting example, themicroRNA incorporated into the nucleic acid of the invention to dampenantigen presentation is miR-142-3p.

In one embodiment, a nucleic acid may be engineered to include microRNAsites which are expressed in different tissues of a subject. As anon-limiting example, a modified nucleic acid, enhanced modified RNA orribonucleic acid of the present invention may be engineered to includemiR-192 and miR-122 to regulate expression of the modified nucleic acid,enhanced modified RNA or ribonucleic acid in the liver and kidneys of asubject. In another embodiment, a modified nucleic acid, enhancedmodified RNA or ribonucleic acid may be engineered to include more thanone microRNA sites for the same tissue. For example, a modified nucleicacid, enhanced modified RNA or ribonucleic acid of the present inventionmay be engineered to include miR-17-92 and miR-126 to regulateexpression of the modified nucleic acid, enhanced modified RNA orribonucleic acid in endothelial cells of a subject.

In one embodiment, the therapeutic window and or differential expressionassociated with the target polypeptide encoded by the modified nucleicacid, enhanced modified RNA or ribonucleic acid encoding a signal (alsoreferred to herein as a polynucleotide) of the invention may be altered.For example, polynucleotides may be designed whereby a death signal ismore highly expressed in cancer cells (or a survival signal in a normalcell) by virtue of the miRNA signature of those cells. Where a cancercell expresses a lower level of a particular miRNA, the polynucleotideencoding the binding site for that miRNA (or miRNAs) would be morehighly expressed. Hence, the target polypeptide encoded by thepolynucleotide is selected as a protein which triggers or induces celldeath. Neighboring noncancer cells, harboring a higher expression of thesame miRNA would be less affected by the encoded death signal as thepolynucleotide would be expressed at a lower level due to the affects ofthe miRNA binding to the binding site or “sensor” encoded in the 3′UTR.Conversely, cell survival or cytoprotective signals may be delivered totissues containing cancer and non cancerous cells where a miRNA has ahigher expression in the cancer cells—the result being a lower survivalsignal to the cancer cell and a larger survival signature to the normalcell. Multiple polynucleotides may be designed and administered havingdifferent signals according to the previous paradigm.

In one embodiment, the expression of a nucleic acid may be controlled byincorporating at least one sensor sequence in the nucleic acid andformulating the nucleic acid. As a non-limiting example, a nucleic acidmay be targeted to an orthotopic tumor by having a nucleic acidincorporating a miR-122 binding site and formulated in a lipidnanoparticle comprising the cationic lipid DLin-KC2-DMA (see e.g., theexperiments described in Example 49A and 49B).

According to the present invention, the polynucleotides may be modifiedas to avoid the deficiencies of other polypeptide-encoding molecules ofthe art. Hence, in this embodiment the polynucleotides are referred toas modified polynucleotides.

Through an understanding of the expression patterns of microRNA indifferent cell types, modified nucleic acids, enhanced modified RNA orribonucleic acids such as polynucleotides can be engineered for moretargeted expression in specific cell types or only under specificbiological conditions. Through introduction of tissue-specific microRNAbinding sites, modified nucleic acids, enhanced modified RNA orribonucleic acids, could be designed that would be optimal for proteinexpression in a tissue or in the context of a biological condition.

Transfection experiments can be conducted in relevant cell lines, usingengineered modified nucleic acids, enhanced modified RNA or ribonucleicacids and protein production can be assayed at various time pointspost-transfection. For example, cells can be transfected with differentmicroRNA binding site-engineering nucleic acids or mRNA and by using anELISA kit to the relevant protein and assaying protein produced at 6 hr,12 hr, 24 hr, 48 hr, 72 hr and 7 days post-transfection. In vivoexperiments can also be conducted using microRNA-binding site-engineeredmolecules to examine changes in tissue-specific expression of formulatedmodified nucleic acids, enhanced modified RNA or ribonucleic acids.

Non-limiting examples of cell lines which may be useful in theseinvestigations include those from ATCC (Manassas, Va.) including MRC-5,A549, T84, NCI-H2126 [H2126], NCI-H1688 [H1688], WI-38, WI-38 VA-13subline 2RA, WI-26 VA4, C3A [HepG2/C3A, derivative of Hep G2 (ATCCHB-8065)], THLE-3, H69AR, NCI-H292 [H292], CFPAC-1, NTERA-2 cl.D1[NT2/D1], DMS 79, DMS 53, DMS 153, DMS 114, MSTO-211H, SW 1573 [SW-1573,SW1573], SW 1271 [SW-1271, SW1271], SHP-77, SNU-398, SNU-449, SNU-182,SNU-475, SNU-387, SNU-423, NL20, NL20-TA [NL20T-A], THLE-2, HBE135-E6E7,HCC827, HCC4006, NCI-H23 [H23], NCI-H1299, NCI-H187 [H187], NCI-H358[H-358, H358], NCI-H378 [H378], NCI-H522 [H522], NCI-H526 [H526],NCI-H727 [H727], NCI-H810 [H810], NCI-H889 [H889], NCI-H1155 [H1155],NCI-H1404 [H1404], NCI-N87 [N87], NCI-H196 [H196], NCI-H211 [H211],NCI-H220 [H220], NCI-H250 [H250], NCI-H524 [H524], NCI-H647 [H647],NCI-H650 [H650], NCI-H711 [H711], NCI-H719 [H719], NCI-H740 [H740],NCI-H748 [H748], NCI-H774 [H774], NCI-H838 [H838], NCI-H841 [H841],NCI-H847 [H847], NCI-H865 [H865], NCI-H920 [H920], NCI-H1048 [H1048],NCI-H1092 [H1092], NCI-H1105 [H1105], NCI-H1184 [H1184], NCI-H1238[H1238], NCI-H1341 [H1341], NCI-H1385 [H1385], NCI-H1417 [H1417],NCI-H1435 [H1435], NCI-H1436 [H1436], NCI-H1437 [H1437], NCI-H1522[H1522], NCI-H1563 [H1563], NCI-H1568 [H1568], NCI-H1573 [H1573],NCI-H1581 [H1581], NCI-H1618 [H1618], NCI-H1623 [H1623], NCI-H1650[H-1650, H1650], NCI-H1651 [H1651], NCI-H1666 [H-1666, H1666], NCI-H1672[H1672], NCI-H1693 [H1693], NCI-H1694 [H1694], NCI-H1703 [H1703],NCI-H1734 [H-1734, H1734], NCI-H1755 [H1755], NCI-H1755 [H1755],NCI-H1770 [H1770], NCI-H1793 [H1793], NCI-H1836 [H1836], NCI-H1838[H1838], NCI-H1869 [H1869], NCI-H1876 [H1876], NCI-H1882 [H1882],NCI-H1915 [H1915], NCI-H1930 [H1930], NCI-H1944 [H1944], NCI-H1975[H-1975, H1975], NCI-H1993 [H1993], NCI-H2023 [H2023], NCI-H2029[H2029], NCI-H2030 [H2030], NCI-H2066 [H2066], NCI-H2073 [H2073],NCI-H2081 [H2081], NCI-H2085 [H2085], NCI-H2087 [H2087], NCI-H2106[H2106], NCI-H2110 [H2110], NCI-H2135 [H2135], NCI-H2141 [H2141],NCI-H2171 [H2171], NCI-H2172 [H2172], NCI-H2195 [H2195], NCI-H2196[H2196], NCI-H2198 [H2198], NCI-H2227 [H2227], NCI-H2228 [H2228],NCI-H2286 [H2286], NCI-H2291 [H2291], NCI-H2330 [H2330], NCI-H2342[H2342], NCI-H2347 [H2347], NCI-H2405 [H2405], NCI-H2444 [H2444],UMC-11, NCI-H64 [H64], NCI-H735 [H735], NCI-H735 [H735], NCI-H1963[H1963], NCI-H2107 [H2107], NCI-H2108 [H2108], NCI-H2122 [H2122], Hs573.T, Hs 573.Lu, PLC/PRF/5, BEAS-2B, Hep G2, Tera-1, Tera-2, NCI-H69[H69], NCI-H128 [H128], ChaGo-K-1, NCI-H446 [H446], NCI-H209 [H209],NCI-H146 [H146], NCI-H441 [H441], NCI-H82 [H82], NCI-H460 [H460],NCI-H596 [H596], NCI-H676B [H676B], NCI-H345 [H345], NCI-H820 [H820],NCI-H520 [H520], NCI-H661 [H661], NCI-H510A [H510A, NCI-H510], SK-HEP-1,A-427, Calu-1, Calu-3, Calu-6, SK-LU-1, SK-MES-1, SW 900 [SW-900,SW900], Malme-3M, and Capan-1.

In some embodiments, modified messenger RNA can be designed toincorporate microRNA binding region sites that either have 100% identityto known seed sequences or have less than 100% identity to seedsequences. The seed sequence can be partially mutated to decreasemicroRNA binding affinity and as such result in reduced downmodulationof that mRNA transcript. In essence, the degree of match or mis-matchbetween the target mRNA and the microRNA seed can act as a rheostat tomore finely tune the ability of the microRNA to modulate proteinexpression. In addition, mutation in the non-seed region of a microRNAbinding site may also impact the ability of a microRNA to modulateprotein expression.

In one embodiment, a miR sequence may be incorporated into the loop of astem loop.

In another embodiment, a miR seed sequence may be incorporated in theloop of a stem loop and a miR binding site may be incorporated into the5′ or 3′ stem of the stem loop.

In one embodiment, a TEE may be incorporated on the 5′ end of the stemof a stem loop and a miR seed may be incorporated into the stem of thestem loop. In another embodiment, a TEE may be incorporated on the 5′end of the stem of a stem loop, a miR seed may be incorporated into thestem of the stem loop and a miR binding site may be incorporated intothe 3′ end of the stem or the sequence after the stem loop. The miR seedand the miR binding site may be for the same and/or different miRsequences.

In one embodiment, the incorporation of a miR sequence and/or a TEEsequence changes the shape of the stem loop region which may increaseand/or decrease translation. (see e.g, Kedde et al. A Pumilio-inducedRNA structure switch in p27-3′UTR controls miR-221 and miR-22accessibility. Nature Cell Biology. 2010, herein incorporated byreference in its entirety).

In one embodiment, the incorporation of a miR sequence and/or a TEEsequence changes the shape of the stem loop region which may increaseand/or decrease translation. (see e.g, Kedde et al. A Pumilio-inducedRNA structure switch in p27-3′UTR controls miR-221 and miR-22accessibility. Nature Cell Biology. 2010, herein incorporated byreference in its entirety).

In one embodiment, the 5′UTR may comprise at least one microRNAsequence. The microRNA sequence may be, but is not limited to, a 19 or22 nucleotide sequence and/or a microRNA sequence without the seed.

In one embodiment the microRNA sequence in the 5′UTR may be used tostabilize the nucleic acid and/or mRNA described herein.

In another embodiment, a microRNA sequence in the 5′UTR may be used todecrease the accessibility of the site of translation initiation suchas, but not limited to a start codon. Matsuda et al (PLoS One. 201011(5):e15057; herein incorporated by reference in its entirety) usedantisense locked nucleic acid (LNA) oligonucleotides and exon-junctioncomplexes (EJCs) around a start codon (−4 to +37 where the A of the AUGcodons is +1) in order to decrease the accessibility to the first startcodon (AUG). Matsuda showed that altering the sequence around the startcodon with an LNA or EJC the efficiency, length and structural stabilityof the nucleic acid or mRNA is affected. The nucleic acids or mRNA ofthe present invention may comprise a microRNA sequence, instead of theLNA or EJC sequence described by Matsuda et al, near the site oftranslation initiation in order to decrease the accessibility to thesite of translation initiation. The site of translation initiation maybe prior to, after or within the microRNA sequence. As a non-limitingexample, the site of translation initiation may be located within amicroRNA sequence such as a seed sequence or binding site. As anothernon-limiting example, the site of translation initiation may be locatedwithin a miR-122 sequence such as the seed sequence or the mir-122binding site.

In one embodiment, the nucleic acids or mRNA of the present inventionmay include at least one microRNA in order to dampen the antigenpresentation by antigen presenting cells. The microRNA may be thecomplete microRNA sequence, the microRNA seed sequence, the microRNAsequence without the seed or a combination thereof. As a non-limitingexample, the microRNA incorporated into the nucleic acids or mRNA of thepresent invention may be specific to the hematopoietic system. Asanother non-limiting example, the microRNA incorporated into the nucleicacids or mRNA of the present invention to dampen antigen presentation ismiR-142-3p.

In one embodiment, the nucleic acids or mRNA of the present inventionmay include at least one microRNA in order to dampen expression of theencoded polypeptide in a cell of interest. As a non-limiting example,the nucleic acids or mRNA of the present invention may include at leastone miR-122 binding site in order to dampen expression of an encodedpolypeptide of interest in the liver. As another non-limiting example,the nucleic acids or mRNA of the present invention may include at leastone miR-142-3p binding site, miR-142-3p seed sequence, miR-142-3pbinding site without the seed, miR-142-5p binding site, miR-142-5p seedsequence, miR-142-5p binding site without the seed, miR-146 bindingsite, miR-146 seed sequence and/or miR-146 binding site without the seedsequence (see e.g., the experiment outlined in Example 24, 25, 26, 26,36 and 48).

In one embodiment, the nucleic acids or mRNA of the present inventionmay comprise at least one microRNA binding site in the 3′UTR in order toselectively degrade mRNA therapeutics in the immune cells to subdueunwanted immunogenic reactions caused by therapeutic delivery. As anon-limiting example, the microRNA binding site may be the modifiednucleic acids more unstable in antigen presenting cells. Non-limitingexamples of these microRNA include mir-142-5p, mir-142-3p, mir-146a-5pand mir-146-3p.

In one embodiment, the nucleic acids or mRNA of the present inventioncomprises at least one microRNA sequence in a region of the nucleic acidor mRNA which may interact with a RNA binding protein.

RNA Motifs for RNA Binding Proteins (RBPs)

RNA binding proteins (RBPs) can regulate numerous aspects of co- andpost-transcription gene expression such as, but not limited to, RNAsplicing, localization, translation, turnover, polyadenylation, capping,modification, export and localization. RNA-binding domains (RBDs), suchas, but not limited to, RNA recognition motif (RR) and hnRNP K-homology(KH) domains, typically regulate the sequence association between RBPsand their RNA targets (Ray et al. Nature 2013. 499:172-177; hereinincorporated by reference in its entirety). In one embodiment, thecanonical RBDs can bind short RNA sequences. In another embodiment, thecanonical RBDs can recognize structure RNAs.

Non limiting examples of RNA binding proteins and related nucleic acidand protein sequences are shown in Table 26 in Example 23.

In one embodiment, to increase the stability of the mRNA of interest, anmRNA encoding HuR can be co-transfected or co-injected along with themRNA of interest into the cells or into the tissue. These proteins canalso be tethered to the mRNA of interest in vitro and then administeredto the cells together. Poly A tail binding protein, PABP interacts witheukaryotic translation initiation factor eIF4G to stimulatetranslational initiation. Co-administration of mRNAs encoding these RBPsalong with the mRNA drug and/or tethering these proteins to the mRNAdrug in vitro and administering the protein-bound mRNA into the cellscan increase the translational efficiency of the mRNA. The same conceptcan be extended to co-administration of mRNA along with mRNAs encodingvarious translation factors and facilitators as well as with theproteins themselves to influence RNA stability and/or translationalefficiency.

In one embodiment, the nucleic acids and/or mRNA may comprise at leastone RNA-binding motif such as, but not limited to a RNA-binding domain(RBD).

In one embodiment, the RBD may be any of the RBDs, fragments or variantsthereof descried by Ray et al. (Nature 2013. 499:172-177; hereinincorporated by reference in its entirety).

In one embodiment, the nucleic acids or mRNA of the present inventionmay comprise a sequence for at least one RNA-binding domain (RBDs). Whenthe nucleic acids or mRNA of the present invention comprise more thanone RBD, the RBDs do not need to be from the same species or even thesame structural class.

In one embodiment, at least one flanking region (e.g., the 5′UTR and/orthe 3′UTR) may comprise at least one RBD. In another embodiment, thefirst flanking region and the second flanking region may both compriseat least one RBD. The RBD may be the same or each of the RBDs may haveat least 60% sequence identity to the other RBD. As a non-limitingexample, at least on RBD may be located before, after and/or within the3′UTR of the nucleic acid or mRNA of the present invention. As anothernon-limiting example, at least one RBD may be located before or withinthe first 300 nucleosides of the 3′UTR.

In another embodiment, the nucleic acids and/or mRNA of the presentinvention may comprise at least one RBD in the first region of linkednucleosides. The RBD may be located before, after or within a codingregion (e.g., the ORF).

In yet another embodiment, the first region of linked nucleosides and/orat least one flanking region may comprise at least on RBD. As anon-limiting example, the first region of linked nucleosides maycomprise a RBD related to splicing factors and at least one flankingregion may comprise a RBD for stability and/or translation factors.

In one embodiment, the nucleic acids and/or mRNA of the presentinvention may comprise at least one RBD located in a coding and/ornon-coding region of the nucleic acids and/or mRNA.

In one embodiment, at least one RBD may be incorporated into at leastone flanking region to increase the stability of the nucleic acid and/ormRNA of the present invention.

In one embodiment, a microRNA sequence in a RNA binding protein motifmay be used to decrease the accessibility of the site of translationinitiation such as, but not limited to a start codon. The nucleic acidsor mRNA of the present invention may comprise a microRNA sequence,instead of the LNA or EJC sequence described by Matsuda et al, near thesite of translation initiation in order to decrease the accessibility tothe site of translation initiation. The site of translation initiationmay be prior to, after or within the microRNA sequence. As anon-limiting example, the site of translation initiation may be locatedwithin a microRNA sequence such as a seed sequence or binding site. Asanother non-limiting example, the site of translation initiation may belocated within a miR-122 sequence such as the seed sequence or themir-122 binding site.

In another embodiment, an antisense locked nucleic acid (LNA)oligonucleotides and exon-junction complexes (EJCs) may be used in theRNA binding protein motif. The LNA and EJCs may be used around a startcodon (−4 to +37 where the A of the AUG codons is +1) in order todecrease the accessibility to the first start codon (AUG).

Other Regulatory Elements in 3′UTR

In addition to microRNA binding sites, other regulatory sequences in the3′-UTR of natural mRNA, which regulate mRNA stability and translation indifferent tissues and cells, can be removed or introduced into modifiedmessenger RNA. Such cis-regulatory elements may include, but are notlimited to, Cis-RNP (Ribonucleoprotein)/RBP (RNA binding protein)regulatory elements, AU-rich element (AUE), structured stem-loop,constitutive decay elements (CDEs), GC-richness and other structuredmRNA motifs (Parker B J et al., Genome Research, 2011, 21, 1929-1943,which is herein incorporated by reference in its entirety). For example,CDEs are a class of regulatory motifs that mediate mRNA degradationthrough their interaction with Roquin proteins. In particular, CDEs arefound in many mRNAs that encode regulators of development andinflammation to limit cytokine production in macrophage (Leppek K etal., 2013, Cell, 153, 869-881, which is herein incorporated by referencein its entirety).

In one embodiment, a particular CDE can be introduced to the nucleicacids or mRNA when the degradation of polypeptides in a cell or tissueis desired. A particular CDE can also be removed from the nucleic acidsor mRNA to maintain a more stable mRNA in a cell or tissue forsustaining protein expression.

Auxotrophic mRNA

In one embodiment, the nucleic acids or mRNA of the present inventionmay be auxotrophic. As used herein, the term “auxotrophic” refers tomRNA that comprises at least one feature that triggers, facilitates orinduces the degradation or inactivation of the mRNA in response tospatial or temporal cues such that protein expression is substantiallyprevented or reduced. Such spatial or temporal cues include the locationof the mRNA to be translated such as a particular tissue or organ orcellular environment. Also contemplated are cues involving temperature,pH, ionic strength, moisture content and the like.

In one embodiment, the feature is located in a terminal region of thenucleic acids or mRNA of the present invention. As a non-limitingexample, the auxotrophic mRNA may contain a miR binding site in theterminal region which binds to a miR expressed in a selected tissue sothat the expression of the auxotrophic mRNA is substantially preventedor reduced in the selected tissue. To this end and for example, anauxotrophic mRNA containing a miR-122 binding site will not produceprotein if localized to the liver since miR-122 is expressed in theliver and binding of the miR would effectuate destruction of theauxotrophic mRNA. As a non-limiting example, HEK293 cells do not expressmiR-122 so there would be little to no downregulation of a nucleic acidor mRNA of the present invention having a miR-122 sequence in HEK293 butfor hepatocytes which do expression miR-122 there would be adownregulation of a nucleic acid or mRNA of the present invention havinga miR-122 sequence in hepatocytes (see e.g., the study outlined Example14). As another non-limiting example, the miR-122 level can be measuredin HeLa cells, primary human hepatocytes and primary rat hepatocytesprior to administration with a nucleic acid or mRNA of the presentinvention encoding at least one miR-122 binding site, miR-122 bindingsite without the seed sequence or a miR-122 binding site Afteradministration the expression of the modified nucleic acid with amicroRNA sequence can be measured to determine the dampening effect ofthe miR-122 in the modified nucleic acid (see e.g., the studies outlinedin Examples 28, 29, 30, 35, 45, 46 and 47). As yet another non-limitingexample, the effectiveness of the miR-122 binding site, miR-122 seed orthe miR-122 binding site without the seed in different 3′UTRs may beevaluated in order to determine the proper UTR for the desired outcomesuch as, but not limited to, the highest dampening effect (see e.g., thestudy outlined in Example 35 and 46).

In one embodiment, the degradation or inactivation of auxotrophic mRNAmay comprise a feature responsive to a change in pH. As a non-limitingexample, the auxotrophic mRNA may be triggered in an environment havinga pH of between pH 4.5 to 8.0 such as at a pH of 5.0 to 6.0 or a pH of6.0 to 6.5. The change in pH may be a change of 0.1 unit, 0.2 units, 0.3units, 0.4 units, 0.5 units, 0.6 units, 0.7 units, 0.8 units, 0.9 units,1.0 units, 1.1 units, 1.2 units, 1.3 units, 1.4 units, 1.5 units, 1.6units, 1.7 units, 1.8 units, 1.9 units, 2.0 units, 2.1 units, 2.2 units,2.3 units, 2.4 units, 2.5 units, 2.6 units, 2.7 units, 2.8 units, 2.9units, 3.0 units, 3.1 units, 3.2 units, 3.3 units, 3.4 units, 3.5 units,3.6 units, 3.7 units, 3.8 units, 3.9 units, 4.0 units or more.

In another embodiment, the degradation or inactivation of auxotrophicmRNA may be triggered or induced by changes in temperature. As anon-limiting example, a change of temperature from room temperature tobody temperature. The change of temperature may be less than 1° C., lessthan 5° C., less than 10° C., less than 15° C., less than 20° C., lessthan 25° C. or more than 25° C.

In yet another embodiment, the degradation or inactivation ofauxotrophic mRNA may be triggered or induced by a change in the levelsof ions in the subject. The ions may be cations or anions such as, butnot limited to, sodium ions, potassium ions, chloride ions, calciumions, magnesium ions and/or phosphate ions.

3′ UTR and the AU Rich Elements

3′UTRs are known to have stretches of Adenosines and Uridines embeddedin them. These AU rich signatures are particularly prevalent in geneswith high rates of turnover. Based on their sequence features andfunctional properties, the AU rich elements (AREs) can be separated intothree classes (Chen et al, 1995): Class I AREs contain several dispersedcopies of an AUUUA motif within U-rich regions. C-Myc and MyoD containclass I AREs. Class II AREs possess two or more overlappingUUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREsinclude GM-CSF and TNF-a. Class III ARES are less well defined. These Urich regions do not contain an AUUUA motif. c-Jun and Myogenin are twowell-studied examples of this class. Most proteins binding to the AREsare known to destabilize the messenger, whereas members of the ELAVfamily, most notably HuR, have been documented to increase the stabilityof mRNA. HuR binds to AREs of all the three classes. Engineering the HuRspecific binding sites into the 3′ UTR of nucleic acid molecules willlead to HuR binding and thus, stabilization of the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of nucleic acids or mRNA of theinvention. When engineering specific nucleic acids or mRNA, one or morecopies of an ARE can be introduced to make nucleic acids or mRNA of theinvention less stable and thereby curtail translation and decreaseproduction of the resultant protein. Likewise, AREs can be identifiedand removed or mutated to increase the intracellular stability and thusincrease translation and production of the resultant protein.Transfection experiments can be conducted in relevant cell lines, usingnucleic acids or mRNA of the invention and protein production can beassayed at various time points post-transfection. For example, cells canbe transfected with different ARE-engineering molecules and by using anELISA kit to the relevant protein and assaying protein produced at 6 hr,12 hr, 24 hr, 48 hr, and 7 days post-transfection.

3′ UTR and Triple Helices

In one embodiment, nucleic acids of the present invention may include atriple helix on the 3′ end of the modified nucleic acid, enhancedmodified RNA or ribonucleic acid. The 3′ end of the nucleic acids of thepresent invention may include a triple helix alone or in combinationwith a Poly-A tail.

In one embodiment, the nucleic acid of the present invention maycomprise at least a first and a second U-rich region, a conserved stemloop region between the first and second region and an A-rich region.The first and second U-rich region and the A-rich region may associateto form a triple helix on the 3′ end of the nucleic acid. This triplehelix may stabilize the nucleic acid, enhance the translationalefficiency of the nucleic acid and/or protect the 3′ end fromdegradation. Exemplary triple helices include, but are not limited to,the triple helix sequence of metastasis-associated lung adenocarcinomatranscript 1 (MALAT1), MEN-β and polyadenylated nuclear (PAN) RNA (SeeWilusz et al., Genes & Development 2012 26:2392-2407; hereinincorporated by reference in its entirety). In one embodiment, the 3′end of the modified nucleic acids, enhanced modified RNA or ribonucleicacids of the present invention comprises a first U-rich regioncomprising TTTTTCTTTT (SEQ ID NO: 1), a second U-rich region comprisingTTTTGCTTTTT (SEQ ID NO: 2) or TTTTGCTTTT (SEQ ID NO: 3), an A-richregion comprising AAAAAGCAAAA (SEQ ID NO: 4). In another embodiment, the3′ end of the nucleic acids of the present invention comprises a triplehelix formation structure comprising a first U-rich region, a conservedregion, a second U-rich region and an A-rich region.

In one embodiment, the triple helix may be formed from the cleavage of aMALAT1 sequence prior to the cloverleaf structure. While not meaning tobe bound by theory, MALAT1 is a long non-coding RNA which, when cleaved,forms a triple helix and a tRNA-like cloverleaf structure. The MALAT1transcript then localizes to nuclear speckles and the tRNA-likecloverleaf localizes to the cytoplasm (Wilusz et al. Cell 2008 135(5):919-932; herein incorporated by reference in its entirety).

As a non-limiting example, the terminal end of the nucleic acid of thepresent invention comprising the MALAT1 sequence can then form a triplehelix structure, after RNaseP cleavage from the cloverleaf structure,which stabilizes the nucleic acid (Peart et al. Non-mRNA 3′ endformation: how the other half lives; WIREs RNA 2013; herein incorporatedby reference in its entirety).

In one embodiment, the nucleic acids or mRNA described herein comprise aMALAT1 sequence. In another embodiment, the nucleic acids or mRNA may bepolyadenylated. In yet another embodiment, the nucleic acids or mRNA isnot polyadenylated but has an increased resistance to degradationcompared to unmodified nucleic acids or mRNA.

In one embodiment, the nucleic acids of the present invention maycomprise a MALAT1 sequence in the second flanking region (e.g., the3′UTR). As a non-limiting example, the MALAT1 sequence may be human ormouse (see e.g., the polynucleotides described in Table 37 in Example38).

In another embodiment, the cloverleaf structure of the MALAT1 sequencemay also undergo processing by RNaseZ and CCA adding enzyme to form atRNA-like structure called mascRNA (MALAT1-associated small cytoplasmicRNA). As a non-limiting example, the mascRNA may encode a protein or afragment thereof and/or may comprise a microRNA sequence. The mascRNAmay comprise at least one chemical modification described herein.

Stem Loop

In one embodiment, the nucleic acids of the present invention mayinclude a stem loop such as, but not limited to, a histone stem loop.The stem loop may be a nucleotide sequence that is about 25 or about 26nucleotides in length such as, but not limited to, SEQ ID NOs: 7-17 asdescribed in International Patent Publication No. WO2013103659, hereinincorporated by reference in its entirety. The histone stem loop may belocated 3′ relative to the coding region (e.g., at the 3′ terminus ofthe coding region). As a non-limiting example, the stem loop may belocated at the 3′ end of a nucleic acid described herein.

In one embodiment, the stem loop may be located in the second terminalregion. As a non-limiting example, the stem loop may be located withinan untranslated region (e.g., 3′UTR) in the second terminal region.

In one embodiment, the nucleic acid such as, but not limited to mRNA,which comprises the histone stem loop may be stabilized by the additionof at least one chain terminating nucleoside. Not wishing to be bound bytheory, the addition of at least one chain terminating nucleoside mayslow the degradation of a nucleic acid and thus can increase thehalf-life of the nucleic acid.

In one embodiment, the chain terminating nucleoside may be, but is notlimited to, those described in International Patent Publication No.WO2013103659, herein incorporated by reference in its entirety. Inanother embodiment, the chain terminating nucleosides which may be usedwith the present invention includes, but is not limited to,3′-deoxyadenosine (cordycepin), 3′-deoxyuridine, 3′-deoxycytosine,3′-deoxyguanosine, 3′-deoxythymine, 2′,3′-dideoxynucleosides, such as2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine,2′,3′-dideoxyguanosine, 2′,3′-dideoxythymine, a 2′-deoxynucleoside, or a—O— methylnucleoside.

In another embodiment, the nucleic acid such as, but not limited tomRNA, which comprises the histone stem loop may be stabilized by amodification to the 3′ region of the nucleic acid that can preventand/or inhibit the addition of oligo(U) (see e.g., International PatentPublication No. WO2013103659, herein incorporated by reference in itsentirety).

In yet another embodiment, the nucleic acid such as, but not limited tomRNA, which comprises the histone stem loop may be stabilized by theaddition of an oligonucleotide that terminates in a 3′-deoxynucleoside,2′,3′-dideoxynucleoside, 3′-0-methylnucleosides, 3′-0-ethylnucleosides,3′-arabinosides, and other modified nucleosides known in the art and/ordescribed herein.

In one embodiment, the nucleic acids of the present invention mayinclude a histone stem loop, a polyA tail sequence and/or a 5′ capstructure. The histone stem loop may be before and/or after the polyAtail sequence. The nucleic acids comprising the histone stem loop and apolyA tail sequence may include a chain terminating nucleoside describedherein.

In another embodiment, the nucleic acids of the present invention mayinclude a histone stem loop and a 5′ cap structure. The 5′ cap structuremay include, but is not limited to, those described herein and/or knownin the art.

In one embodiment, the conserved stem loop region may comprise a miRsequence described herein. As a non-limiting example, the stem loopregion may comprise the seed sequence of a miR sequence describedherein. In another non-limiting example, the stem loop region maycomprise a miR-122 seed sequence.

In another embodiment, the conserved stem loop region may comprise a miRsequence described herein and may also include a TEE sequence.

In one embodiment, the incorporation of a miR sequence and/or a TEEsequence changes the shape of the stem loop region which may increaseand/or decrease translation. (see e.g, Kedde et al. A Pumilio-inducedRNA structure switch in p27-3′UTR controls miR-221 and miR-22accessibility. Nature Cell Biology. 2010, herein incorporated byreference in its entirety).

In one embodiment, the modified nucleic acids described herein maycomprise at least one histone stem-loop and a polyA sequence orpolyadenylation signal. Non-limiting examples of nucleic acid sequencesencoding for at least one histone stem-loop and a polyA sequence or apolyadenylation signal are described in International Patent PublicationNo. WO2013120497, WO2013120629, WO2013120500, WO2013120627,WO2013120498, WO2013120626, WO2013120499 and WO2013120628, the contentsof each of which is herein incorporated by reference in their entirety.In one embodiment, the nucleic acid encoding for a histone stem loop anda polyA sequence or a polyadenylation signal may code for a pathogenantigen or fragment thereof such as the nucleic acid sequences describedin International Patent Publication No WO2013120499 and WO2013120628,the contents of which is herein incorporated by reference in itsentirety. In another embodiment, the nucleic acid encoding for a histonestem loop and a polyA sequence or a polyadenylation signal may code fora therapeutic protein such as the nucleic acid sequences described inInternational Patent Publication No WO2013120497 and WO2013120629, thecontents of which is herein incorporated by reference in its entirety.In one embodiment, the nucleic acid encoding for a histone stem loop anda polyA sequence or a polyadenylation signal may code for a tumorantigen or fragment thereof such as the nucleic acid sequences describedin International Patent Publication No WO2013120500 and WO2013120627,the contents of which is herein incorporated by reference in itsentirety. In another embodiment, the nucleic acid encoding for a histonestem loop and a polyA sequence or a polyadenylation signal may code fora allergenic antigen or an autoimmune self-antigen such as the nucleicacid sequences described in International Patent Publication NoWO2013120498 and WO2013120626, the contents of which is hereinincorporated by reference in its entirety.

5′ Capping

The 5′ cap structure of an mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA. This5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

Modifications to the nucleic acids of the present invention may generatea non-hydrolyzable cap structure preventing decapping and thusincreasing mRNA half-life. Because cap structure hydrolysis requirescleavage of 5′-ppp-5′ phosphorodiester linkages, modified nucleotidesmay be used during the capping reaction. For example, a Vaccinia CappingEnzyme from New England Biolabs (Ipswich, Mass.) may be used withα-thio-guanosine nucleotides according to the manufacturer'sinstructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap.Additional modified guanosine nucleotides may be used such asα-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the mRNA (as mentioned above) on the2′-hydroxyl group of the sugar ring. Multiple distinct 5′-cap structurescan be used to generate the 5′-cap of a nucleic acid molecule, such asan mRNA molecule.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e. endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e. non-enzymatically) orenzymatically synthesized and/linked to a nucleic acid molecule.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′ mppp-G;which may equivalently be designated 3′ O-Me-m7G(5′)ppp(5′)G). The 3′-Oatom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA).The N7- and 3′-O-methylated guanine provides the terminal moiety of thecapped nucleic acid molecule (e.g. mRNA or mmRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-β-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

In one embodiment, the cap is a dinucleotide cap analog. As anon-limiting example, the dinucleotide cap analog may be modified atdifferent phosphate positions with a boranophosphate group or aphosphoroselenoate group such as the dinucleotide cap analogs describedin U.S. Pat. No. 8,519,110, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, the cap is a cap analog is aN7-(4-chlorophenoxyethyl) substituted dicucleotide form of a cap analogknown in the art and/or described herein. Non-limiting examples of aN7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analoginclude a N7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G and aN7-(4-chlorophenoxyethyl)-m^(3′-O)G(5′)ppp(5′)G cap analog (See e.g.,the various cap analogs and the methods of synthesizing cap analogsdescribed in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of which are herein incorporated by referencein its entirety). In another embodiment, a cap analog of the presentinvention is a 4-chloro/bromophenoxyethyl analog.

While cap analogs allow for the concomitant capping of a nucleic acidmolecule in an in vitro transcription reaction, up to 20% of transcriptsremain uncapped. This, as well as the structural differences of a capanalog from an endogenous 5′-cap structures of nucleic acids produced bythe endogenous, cellular transcription machinery, may lead to reducedtranslational competency and reduced cellular stability.

Modified nucleic acids of the invention may also be cappedpost-transcriptionally, using enzymes, in order to generate moreauthentic 5′-cap structures. As used herein, the phrase “more authentic”refers to a feature that closely mirrors or mimics, either structurallyor functionally, an endogenous or wild type feature. That is, a “moreauthentic” feature is better representative of an endogenous, wild-type,natural or physiological cellular function and/or structure as comparedto synthetic features or analogs, etc., of the prior art, or whichoutperforms the corresponding endogenous, wild-type, natural orphysiological feature in one or more respects. Non-limiting examples ofmore authentic 5′ cap structures of the present invention are thosewhich, among other things, have enhanced binding of cap bindingproteins, increased half life, reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping, as compared to synthetic 5′cap structures known in the art (or to a wild-type, natural orphysiological 5′ cap structure). For example, recombinant Vaccinia VirusCapping Enzyme and recombinant 2′-O-methyltransferase enzyme can createa canonical 5′-5′-triphosphate linkage between the 5′-terminalnucleotide of an mRNA and a guanine cap nucleotide wherein the capguanine contains an N7 methylation and the 5′-terminal nucleotide of themRNA contains a 2′-O-methyl. Such a structure is termed the Cap1structure. This cap results in a higher translational-competency andcellular stability and a reduced activation of cellular pro-inflammatorycytokines, as compared, e.g., to other 5′ cap analog structures known inthe art. Cap structures include 7mG(5′)ppp(5′)N,pN2p (cap 0),7mG(5′)ppp(5′)NlmpNp (cap 1), 7mG(5′)-ppp(5′)NlmpN2 mp (cap 2) andm(7)Gpppm(3)(6,6,2′)Apm(2′)Apm(2′)Cpm(2)(3,2′)Up (cap 4).

Because the modified nucleic acids may be capped post-transcriptionally,and because this process is more efficient, nearly 100% of the modifiednucleic acids may be capped. This is in contrast to ˜80% when a capanalog is linked to an mRNA in the course of an in vitro transcriptionreaction.

According to the present invention, 5′ terminal caps may includeendogenous caps or cap analogs. According to the present invention, a 5′terminal cap may comprise a guanine analog. Useful guanine analogsinclude inosine, N1-methyl-guanosine, 2′ fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,and 2-azido-guanosine.

In one embodiment, the nucleic acids described herein may contain amodified 5′ cap. A modification on the 5′ cap may increase the stabilityof mRNA, increase the half-life of the mRNA, and could increase the mRNAtranslational efficiency. The modified 5′ cap may include, but is notlimited to, one or more of the following modifications: modification atthe 2′ and/or 3′ position of a capped guanosine triphosphate (GTP), areplacement of the sugar ring oxygen (that produced the carbocyclicring) with a methylene moiety (CH₂), a modification at the triphosphatebridge moiety of the cap structure, or a modification at the nucleobase(G) moiety.

The 5′ cap structure that may be modified includes, but is not limitedto, the caps described herein such as Cap0 having the substratestructure for cap dependent translation of:

or Cap1 having the substrate structure for cap dependent translation of:

As a non-limiting example, the modified 5′ cap may have the substratestructure for cap dependent translation of:

where R₁ and R₂ are defined in Table 1:

TABLE 1 Cap Structure Number R₁ R₂ CAP-022 C₂H₅ (Ethyl) H CAP-023 H C₂H₅(Ethyl) CAP-024 C₂H₅ (Ethyl) C₂H₅ (Ethyl) CAP-025 C₃H₇ (Propyl) HCAP-026 H C₃H₇ (Propyl) CAP-027 C₃H₇ (Propyl) C₃H₇ (Propyl) CAP-028 C₄H₉(Butyl) H CAP-029 H C₄H₉ (Butyl) CAP-030 C₄H₉ (Butyl) C₄H₉ (Butyl)CAP-031 C₅H₁₁ (Pentyl) H CAP-032 H C₅H₁₁ (Pentyl) CAP-033 C₅H₁₁ (Pentyl)C₅H₁₁ (Pentyl) CAP-034 H₂C—C≡CH (Propargyl) H CAP-035 H H₂C—C≡CH(Propargyl) CAP-036 H₂C—C≡CH (Propargyl) H₂C—C≡CH (Propargyl) CAP-037CH₂CH═CH₂ (Allyl) H CAP-038 H CH₂CH═CH₂ (Allyl) CAP-039 CH₂CH═CH₂(Allyl) CH₂CH═CH₂ (Allyl) CAP-040 CH₂OCH₃ (MOM) H CAP-041 H CH₂OCH₃(MOM) CAP-042 CH₂OCH₃ (MOM) CH₂OCH₃ (MOM) CAP-043 CH₂OCH₂CH₂OCH₃ (MEM) HCAP-044 H CH₂OCH₂CH₂OCH₃ (MEM) CAP-045 CH₂OCH₂CH₂OCH₃ (MEM)CH₂OCH₂CH₂OCH₃ (MEM) CAP-046 CH₂SCH₃ (MTM) H CAP-047 H CH₂SCH₃ (MTM)CAP-048 CH₂SCH₃ (MTM) CH₂SCH₃ (MTM) CAP-049 CH₂C₆H₅ (Benzyl) H CAP-050 HCH₂C₆H₅ (Benzyl) CAP-051 CH₂C₆H₅ (Benzyl) CH₂C₆H₅ (Benzyl) CAP-052CH₂OCH₂C₆H₅ (BOM) H CAP-053 H CH₂OCH₂C₆H₅ (BOM) CAP-054 CH₂OCH₂C₆H₅(BOM) CH₂OCH₂C₆H₅ (BOM) CAP-055 CH₂C₆H₄—OMe (p- H Methoxybenzyl) CAP-056H CH₂C₆H₄—OMe (p-Methoxybenzyl) CAP-057 CH₂C₆H₄—OMe (p- CH₂C₆H₄—OMe(p-Methoxybenzyl) Methoxybenzyl) CAP-058 CH₂C₆H₄—NO₂ (p-Nitrobenzyl) HCAP-059 H CH₂C₆H₄—NO₂ (p-Nitrobenzyl) CAP-060 CH₂C₆H₄—NO₂(p-Nitrobenzyl) CH₂C₆H₄—NO₂ (p-Nitrobenzyl) CAP-061 CH₂C₆H₄—X(p-Halobenzyl) H where X═F, Cl, Br or I CAP-062 H CH₂C₆H₄—X(p-Halobenzyl) where X═F, Cl, Br or I CAP-063 CH₂C₆H₄—X (p-Halobenzyl)CH₂C₆H₄—X (p-Halobenzyl) where where X═F, Cl, Br or I X═F, Cl, Br or ICAP-064 CH₂C₆H₄—N₃ (p-Azidobenzyl) H CAP-065 H CH₂C₆H₄—N₃(p-Azidobenzyl) CAP-066 CH₂C₆H₄—N₃ (p-Azidobenzyl) CH₂C₆H₄—N₃(p-Azidobenzyl) CAP-067 CH₂C₆H₄—CF₃ (p- H Trifluoromethylbenzyl) CAP-068H CH₂C₆H₄—CF₃ (p- Trifluoromethylbenzyl) CAP-069 CH₂C₆H₄—CF₃ (p-CH₂C₆H₄—CF₃ (p- Trifluoromethylbenzyl) Trifluoromethylbenzyl) CAP-070CH₂C₆H₄—OCF₃ (p- H Trifluoromethoxylbenzyl) CAP-071 H CH₂C₆H₄—OCF₃ (p-Trifluoromethoxylbenzyl) CAP-072 CH₂C₆H₄—OCF₃ (p- CH₂C₆H₄—OCF₃ (p-Trifluoromethoxylbenzyl) Trifluoromethoxylbenzyl) CAP-073 CH₂C₆H₃—(CF₃)₂[2,4- H bis(Trifluoromethyl)benzyl] CAP-074 H CH₂C₆H₃—(CF₃)₂ [2,4-bis(Trifluoromethyl)benzyl] CAP-075 CH₂C₆H₃—(CF₃)₂ [2,4- CH₂C₆H₃—(CF₃)₂[2,4- bis(Trifluoromethyl)benzyl] bis(Trifluoromethyl)benzyl] CAP-076Si(C₆H₅)₂C₄H₉ (t- H Butyldiphenylsilyl) CAP-077 H Si(C₆H₅)₂C₄H₉ (t-Butyldiphenylsilyl) CAP-078 Si(C₆H₅)₂C₄H₉ (t- Si(C₆H₅)₂C₄H₉ (t-Butyldiphenylsilyl) Butyldiphenylsilyl) CAP-079 CH₂CH₂CH═CH₂ (Homoallyl)H CAP-080 H CH₂CH₂CH═CH₂ (Homoallyl) CAP-081 CH₂CH₂CH═CH₂ (Homoallyl)CH₂CH₂CH═CH₂ (Homoallyl) CAP-082 P(O)(OH)₂ (MP) H CAP-083 H P(O)(OH)₂(MP) CAP-084 P(O)(OH)₂ (MP) P(O)(OH)₂ (MP) CAP-085 P(S)(OH)₂ (Thio-MP) HCAP-086 H P(S)(OH)₂ (Thio-MP) CAP-087 P(S)(OH)₂ (Thio-MP) P(S)(OH)₂(Thio-MP) CAP-088 P(O)(CH₃)(OH) H (Methylphophonate) CAP-089 HP(O)(CH₃)(OH) (Methylphophonate) CAP-090 P(O)(CH₃)(OH) P(O)(CH₃)(OH)(Methylphophonate) (Methylphophonate) CAP-091 PN(^(i)Pr)₂(OCH₂CH₂CN) H(Phosporamidite) CAP-092 H PN(^(i)Pr)₂(OCH₂CH₂CN) (Phosporamidite)CAP-093 PN(^(i)Pr)₂(OCH₂CH₂CN) PN(^(i)Pr)₂(OCH₂CH₂CN) (Phosporamidite)(Phosporamidite) CAP-094 SO₂CH₃ (Methanesulfonic acid) H CAP-095 HSO₂CH₃ (Methanesulfonic acid) CAP-096 SO₂CH₃ (Methanesulfonic acid)SO₂CH₃ (Methanesulfonic acid)or

where R₁ and R₂ are defined in Table 2:

TABLE 2 Cap Structure Number R1 R2 CAP-097 NH₂ (amino) H CAP-098 H NH₂(amino) CAP-099 NH₂ (amino) NH₂ (amino) CAP-100 N₃ (Azido) H CAP-101 HN₃ (Azido) CAP-102 N₃ (Azido) N₃ (Azido) CAP-103 X (Halo: F, Cl, Br, I)H CAP-104 H X (Halo: F, Cl, Br, I) CAP-105 X (Halo: F, Cl, Br, I) X(Halo: F, Cl, Br, I) CAP-106 SH (Thiol) H CAP-107 H SH (Thiol) CAP-108SH (Thiol) SH (Thiol) CAP-109 SCH₃ (Thiomethyl) H CAP-110 H SCH₃(Thiomethyl) CAP-111 SCH₃ (Thiomethyl) SCH₃ (Thiomethyl)

In Table 1, “MOM” stands for methoxymethyl, “MEM” stands formethoxyethoxymethyl, “MTM” stands for methylthiomethyl, “BOM” stands forbenzyloxymethyl and “MP” stands for monophosphonate. In Table 1 and 2,“F” stands for fluorine, “Cl” stands for chlorine, “Br” stands forbromine and “I” stands for iodine.

In a non-limiting example, the modified 5′ cap may have the substratestructure for vaccinia mRNA capping enzyme of:

where R₁ and R₂ are defined in Table 3:

TABLE 3 Cap Structure Number R₁ R₂ CAP-136 C₂H₅ (Ethyl) H CAP-137 H C₂H₅(Ethyl) CAP-138 C₂H₅ (Ethyl) C₂H₅ (Ethyl) CAP-139 C₃H₇ (Propyl) HCAP-140 H C₃H₇ (Propyl) CAP-141 C₃H₇ (Propyl) C₃H₇ (Propyl) CAP-142 C₄H₉(Butyl) H CAP-143 H C₄H₉ (Butyl) CAP-144 C₄H₉ (Butyl) C₄H₉ (Butyl)CAP-145 C₅H₁₁ (Pentyl) H CAP-146 H C₅H₁₁ (Pentyl) CAP-147 C₅H₁₁ (Pentyl)C₅H₁₁ (Pentyl) CAP-148 H₂C—C≡CH (Propargyl) H CAP-149 H H₂C—C≡CH(Propargyl) CAP-150 H₂C—C≡CH (Propargyl) H₂C—C≡CH (Propargyl) CAP-151CH₂CH═CH₂ (Allyl) H CAP-152 H CH₂CH═CH₂ (Allyl) CAP-153 CH₂CH═CH₂(Allyl) CH₂CH═CH₂ (Allyl) CAP-154 CH₂OCH₃ (MOM) H CAP-155 H CH₂OCH₃(MOM) CAP-156 CH₂OCH₃ (MOM) CH₂OCH₃ (MOM) CAP-157 CH₂OCH₂CH₂OCH₃ (MEM) HCAP-158 H CH₂OCH₂CH₂OCH₃ (MEM) CAP-159 CH₂OCH₂CH₂OCH₃ (MEM)CH₂OCH₂CH₂OCH₃ (MEM) CAP-160 CH₂SCH₃ (MTM) H CAP-161 H CH₂SCH₃ (MTM)CAP-162 CH₂SCH₃ (MTM) CH₂SCH₃ (MTM) CAP-163 CH₂C₆H₅ (Benzyl) H CAP-164 HCH₂C₆H₅ (Benzyl) CAP-165 CH₂C₆H₅ (Benzyl) CH₂C₆H₅ (Benzyl) CAP-166CH₂OCH₂C₆H₅ (BOM) H CAP-167 H CH₂OCH₂C₆H₅ (BOM) CAP-168 CH₂OCH₂C₆H₅(BOM) CH₂OCH₂C₆H₅ (BOM) CAP-169 CH₂C₆H₄—OMe (p- H Methoxybenzyl) CAP-170H CH₂C₆H₄—OMe (p-Methoxybenzyl) CAP-171 CH₂C₆H₄—OMe (p- CH₂C₆H₄—OMe(p-Methoxybenzyl) Methoxybenzyl) CAP-172 CH₂C₆H₄—NO₂ (p- H Nitrobenzyl)CAP-173 H CH₂C₆H₄—NO₂ (p-Nitrobenzyl) CAP-174 CH₂C₆H₄—NO₂ (p-CH₂C₆H₄—NO₂ (p-Nitrobenzyl) Nitrobenzyl) CAP-175 CH₂C₆H₄—X(p-Halobenzyl) H where X═F, Cl, Br or I CAP-176 H CH₂C₆H₄—X(p-Halobenzyl) where X═F, Cl, Br or I CAP-177 CH₂C₆H₄—X (p-Halobenzyl)CH₂C₆H₄—X (p-Halobenzyl) where where X═F, Cl, Br or I X═F, Cl, Br or ICAP-178 CH₂C₆H₄—N₃ (p-Azidobenzyl) H CAP-179 H CH₂C₆H₄—N₃(p-Azidobenzyl) CAP-180 CH₂C₆H₄—N₃ (p-Azidobenzyl) CH₂C₆H₄—N₃(p-Azidobenzyl) CAP-181 CH₂C₆H₄—CF₃ (p- H Trifluoromethylbenzyl) CAP-182H CH₂C₆H₄—CF₃ (p- Trifluoromethylbenzyl) CAP-183 CH₂C₆H₄—CF₃ (p-CH₂C₆H₄—CF₃ (p- Trifluoromethylbenzyl) Trifluoromethylbenzyl) CAP-184CH₂C₆H₄—OCF₃ (p- H Trifluoromethoxylbenzyl) CAP-185 H CH₂C₆H₄—OCF₃ (p-Trifluoromethoxylbenzyl) CAP-186 CH₂C₆H₄—OCF₃ (p- CH₂C₆H₄—OCF₃ (p-Trifluoromethoxylbenzyl) Trifluoromethoxylbenzyl) CAP-187 CH₂C₆H₃—(CF₃)₂[2,4- H bis(Trifluoromethyl)benzyl] CAP-188 H CH₂C₆H₃—(CF₃)₂ [2,4-bis(Trifluoromethyl)benzyl] CAP-189 CH₂C₆H₃—(CF₃)₂ [2,4- CH₂C₆H₃—(CF₃)₂[2,4- bis(Trifluoromethyl)benzyl] bis(Trifluoromethyl)benzyl] CAP-190Si(C₆H₅)₂C₄H₉ (t- H Butyldiphenylsilyl) CAP-191 H Si(C₆H₅)₂C₄H₉(t-Butyldiphenylsilyl) CAP-192 Si(C₆H₅)₂C₄H₉ (t- Si(C₆H₅)₂C₄H₉(t-Butyldiphenylsilyl) Butyldiphenylsilyl) CAP-193 CH₂CH₂CH═CH₂ H(Homoallyl) CAP-194 H CH₂CH₂CH═CH₂ (Homoallyl) CAP-195 CH₂CH₂CH═CH₂CH₂CH₂CH═CH₂ (Homoallyl) (Homoallyl) CAP-196 P(O)(OH)₂ (MP) H CAP-197 HP(O)(OH)₂ (MP) CAP-198 P(O)(OH)₂ (MP) P(O)(OH)₂ (MP) CAP-199 P(S)(OH)₂(Thio-MP) H CAP-200 H P(S)(OH)₂ (Thio-MP) CAP-201 P(S)(OH)₂ (Thio-MP)P(S)(OH)₂ (Thio-MP) CAP-202 P(O)(CH₃)(OH) H (Methylphophonate) CAP-203 HP(O)(CH₃)(OH) (Methylphophonate) CAP-204 P(O)(CH₃)(OH) P(O)(CH₃)(OH)(Methylphophonate) (Methylphophonate) CAP-205 PN(^(i)Pr)₂(OCH₂CH₂CN) H(Phosporamidite) CAP-206 H PN(^(i)Pr)₂(OCH₂CH₂CN) (Phosporamidite)CAP-207 PN(^(i)Pr)₂(OCH₂CH₂CN) PN(^(i)Pr)₂(OCH₂CH₂CN) (Phosporamidite)(Phosporamidite) CAP-208 SO₂CH₃ (Methanesulfonic H acid) CAP-209 HSO₂CH₃ (Methanesulfonic acid) CAP-210 SO₂CH₃ (Methanesulfonic SO₂CH₃(Methanesulfonic acid) acid)or

where R₁ and R₂ are defined in Table 4:

TABLE 4 Cap Structure Number R1 R2 CAP-211 NH₂ (amino) H CAP-212 H NH₂(amino) CAP-213 NH₂ (amino) NH₂ (amino) CAP-214 N₃ (Azido) H CAP-215 HN₃ (Azido) CAP-216 N₃ (Azido) N₃ (Azido) CAP-217 X (Halo: F, Cl, Br, I)H CAP-218 H X (Halo: F, Cl, Br, I) CAP-219 X (Halo: F, Cl, Br, I) X(Halo: F, Cl, Br, I) CAP-220 SH (Thiol) H CAP-221 H SH (Thiol) CAP-222SH (Thiol) SH (Thiol) CAP-223 SCH₃ (Thiomethyl) H CAP-224 H SCH₃(Thiomethyl) CAP-225 SCH₃ (Thiomethyl) SCH₃ (Thiomethyl)

In Table 3, “MOM” stands for methoxymethyl, “MEM” stands formethoxyethoxymethyl, “MTM” stands for methylthiomethyl, “BOM” stands forbenzyloxymethyl and “MP” stands for monophosphonate. In Table 3 and 4,“F” stands for fluorine, “Cl” stands for chlorine, “Br” stands forbromine and “I” stands for iodine.

In another non-limiting example, of the modified capping structuresubstrates CAP-112-CAP-225 could be added in the presence of vacciniacapping enzyme with a component to create enzymatic activity such as,but not limited to, S-adenosylmethionine (AdoMet), to form a modifiedcap for mRNA.

In one embodiment, the replacement of the sugar ring oxygen (thatproduced the carbocyclic ring) with a methylene moiety (CH₂) couldcreate greater stability to the C—N bond against phosphorylases as theC—N bond is resitant to acid or enzymatic hydrolysis. The methylenemoiety may also increase the stability of the triphosphate bridge moietyand thus increasing the stability of the mRNA. As a non-limitingexample, the cap substrate structure for cap dependent translation mayhave the structure such as, but not limited to, CAP-014 and CAP-015and/or the cap substrate structure for vaccinia mRNA capping enzyme suchas, but not limited to, CAP-123 and CAP-124. In another example,CAP-112-CAP-122 and/or CAP-125-CAP-225, can be modified by replacing thesugar ring oxygen (that produced the carbocyclic ring) with a methylenemoiety (CH₂).

In another embodiment, the triphophosphate bridge may be modified by thereplacement of at least one oxygen with sulfur (thio), a borane (BH₃)moiety, a methyl group, an ethyl group, a methoxy group and/orcombinations thereof. This modification could increase the stability ofthe mRNA towards decapping enzymes. As a non-limiting example, the capsubstrate structure for cap dependent translation may have the structuresuch as, but not limited to, CAP-016-CAP-021 and/or the cap substratestructure for vaccinia mRNA capping enzyme such as, but not limited to,CAP-125-CAP-130. In another example, CAP-003-CAP-015, CAP-022-CAP-124and/or CAP-131-CAP-225, can be modified on the triphosphate bridge byreplacing at least one of the triphosphate bridge oxygens with sulfur(thio), a borane (BH₃) moiety, a methyl group, an ethyl group, a methoxygroup and/or combinations thereof.

In one embodiment, CAP-001-134 and/or CAP-136-CAP-225 may be modified tobe a thioguanosine analog similar to CAP-135. The thioguanosine analogmay comprise additional modifications such as, but not limited to, amodification at the triphosphate moiety (e.g., thio, BH₃, CH₃, C₂H₅,OCH₃, S and S with OCH₃), a modification at the 2′ and/or 3′ positionsof 6-thio guanosine as described herein and/or a replacement of thesugar ring oxygen (that produced the carbocyclic ring) as describedherein.

In one embodiment, CAP-001-121 and/or CAP-123-CAP-225 may be modified tobe a modified 5′ cap similar to CAP-122. The modified 5′ cap maycomprise additional modifications such as, but not limited to, amodification at the triphosphate moiety (e.g., thio, BH₃, CH₃, C₂H₅,OCH₃, S and S with OCH₃), a modification at the 2′ and/or 3′ positionsof 6-thio guanosine as described herein and/or a replacement of thesugar ring oxygen (that produced the carbocyclic ring) as describedherein.

In one embodiment, the 5′ cap modification may be the attachment ofbiotin or conjugation at the 2′ or 3′ position of a GTP.

In another embodiment, the 5′ cap modification may include a CF₂modified triphosphate moiety.

3′ UTR and Viral Sequences

Additional viral sequences such as, but not limited to, the translationenhancer sequence of the barley yellow dwarf virus (BYDV-PAV) can beengineered and inserted in the 3′ UTR of the nucleic acids or mRNA ofthe invention and can stimulate the translation of the construct invitro and in vivo. Transfection experiments can be conducted in relevantcell lines at and protein production can be assayed by ELISA at 12 hr,24 hr, 48 hr, 72 hr and day 7 post-transfection.

IRES Sequences

Further, provided are nucleic acids containing an internal ribosomeentry site (IRES). First identified as a feature Picorna virus RNA, IRESplays an important role in initiating protein synthesis in absence ofthe 5′ cap structure. An IRES may act as the sole ribosome binding site,or may serve as one of multiple ribosome binding sites of an mRNA.Nucleic acids or mRNA containing more than one functional ribosomebinding site may encode several peptides or polypeptides that aretranslated independently by the ribosomes (“multicistronic nucleic acidmolecules”). When nucleic acids or mRNA are provided with an IRES,further optionally provided is a second translatable region. Examples ofIRES sequences that can be used according to the invention includewithout limitation, those from picornaviruses (e.g. FMDV), pest viruses(CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV),foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV),classical swine fever viruses (CSFV), murine leukemia virus (MLV),simian immune deficiency viruses (SIV) or cricket paralysis viruses(CrPV).

Terminal Architecture Modifications: Poly-A Tails

During RNA processing, a long chain of adenine nucleotides (poly-A tail)is normally added to a messenger RNA (mRNA) molecules to increase thestability of the molecule. Immediately after transcription, the 3′ endof the transcript is cleaved to free a 3′ hydroxyl. Then poly-Apolymerase adds a chain of adenine nucleotides to the RNA. The process,called polyadenylation, adds a poly-A tail that is between 100 and 250residues long.

It has been discovered that unique poly-A tail lengths provide certainadvantages to the modified RNAs of the present invention.

Generally, the length of a poly-A tail of the present invention isgreater than 30 nucleotides in length. In another embodiment, the poly-Atail is greater than 35 nucleotides in length. In another embodiment,the length is at least 40 nucleotides. In another embodiment, the lengthis at least 45 nucleotides. In another embodiment, the length is atleast 55 nucleotides. In another embodiment, the length is at least 60nucleotides. In another embodiment, the length is at least 60nucleotides. In another embodiment, the length is at least 80nucleotides. In another embodiment, the length is at least 90nucleotides. In another embodiment, the length is at least 100nucleotides. In another embodiment, the length is at least 120nucleotides. In another embodiment, the length is at least 140nucleotides. In another embodiment, the length is at least 160nucleotides. In another embodiment, the length is at least 180nucleotides. In another embodiment, the length is at least 200nucleotides. In another embodiment, the length is at least 250nucleotides. In another embodiment, the length is at least 300nucleotides. In another embodiment, the length is at least 350nucleotides. In another embodiment, the length is at least 400nucleotides. In another embodiment, the length is at least 450nucleotides. In another embodiment, the length is at least 500nucleotides. In another embodiment, the length is at least 600nucleotides. In another embodiment, the length is at least 700nucleotides. In another embodiment, the length is at least 800nucleotides. In another embodiment, the length is at least 900nucleotides. In another embodiment, the length is at least 1000nucleotides. In another embodiment, the length is at least 1100nucleotides. In another embodiment, the length is at least 1200nucleotides. In another embodiment, the length is at least 1300nucleotides. In another embodiment, the length is at least 1400nucleotides. In another embodiment, the length is at least 1500nucleotides. In another embodiment, the length is at least 1600nucleotides. In another embodiment, the length is at least 1700nucleotides. In another embodiment, the length is at least 1800nucleotides. In another embodiment, the length is at least 1900nucleotides. In another embodiment, the length is at least 2000nucleotides. In another embodiment, the length is at least 2500nucleotides. In another embodiment, the length is at least 3000nucleotides.

In some embodiments, the nucleic acid or mRNA includes from about 30 toabout 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500,from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000,from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500,from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500to 3,000).

In one embodiment, the poly-A tail may be 80 nucleotides, 120nucleotides, 160 nucleotides in length on a modified RNA moleculedescribed herein such as, but not limited to, the polyA tail length onthe modified RNA described in Example 13.

In another embodiment, the poly-A tail may be 20, 40, 80, 100, 120, 140or 160 nucleotides in length on a modified RNA molecule described hereinsuch as, but not limited to, the polyA tail length on the modified RNAdescribed in Example 44.

In one embodiment, the poly-A tail is designed relative to the length ofthe overall modified RNA molecule. This design may be based on thelength of the coding region of the modified RNA, the length of aparticular feature or region of the modified RNA (such as the mRNA), orbased on the length of the ultimate product expressed from the modifiedRNA. When relative to any additional feature of the modified RNA (e.g.,other than the mRNA portion which includes the poly-A tail) the poly-Atail may be 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% greater in lengththan the additional feature. The poly-A tail may also be designed as afraction of the modified RNA to which it belongs. In this context, thepoly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of thetotal length of the construct or the total length of the construct minusthe poly-A tail.

In one embodiment, engineered binding sites and/or the conjugation ofnucleic acids or mRNA for Poly-A binding protein may be used to enhanceexpression. The engineered binding sites may be sensor sequences whichcan operate as binding sites for ligands of the local microenvironmentof the nucleic acids and/or mRNA. As a non-limiting example, the nucleicacids and/or mRNA may comprise at least one engineered binding site toalter the binding affinity of Poly-A binding protein (PABP) and analogsthereof. The incorporation of at least one engineered binding site mayincrease the binding affinity of the PABP and analogs thereof.

Additionally, multiple distinct nucleic acids or mRNA may be linkedtogether to the PABP (Poly-A binding protein) through the 3′-end usingmodified nucleotides at the 3′-terminus of the poly-A tail. Transfectionexperiments can be conducted in relevant cell lines at and proteinproduction can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day7 post-transfection. As a non-limiting example, the transfectionexperiments may be used to evaluate the effect on PABP or analogsthereof binding affinity as a result of the addition of at least oneengineered binding site.

In one embodiment, a polyA tail may be used to modulate translationinitiation. While not wishing to be bound by theory, the polyA tailrecruits PABP which in turn can interact with translation initiationcomplex and thus may be essential for protein synthesis.

In another embodiment, a polyA tail may also be used in the presentinvention to protect against 3′-5′ exonuclease digestion.

In one embodiment, the nucleic acids or mRNA of the present inventionare designed to include a polyA-G Quartet. The G-quartet is a cyclichydrogen bonded array of four guanine nucleotides that can be formed byG-rich sequences in both DNA and RNA. In this embodiment, the G-quartetis incorporated at the end of the poly-A tail. The resultant nucleicacid or mRNA may be assayed for stability, protein production and otherparameters including half-life at various time points. It has beendiscovered that the polyA-G quartet results in protein productionequivalent to at least 75% of that seen using a poly-A tail of 120nucleotides alone.

In one embodiment, the nucleic acids or mRNA of the present inventionmay comprise a polyA tail and may be stabilized by the addition of achain terminating nucleoside. The nucleic acids and/or mRNA with a polyAtail may further comprise a 5′ cap structure.

In another embodiment, the nucleic acids or mRNA of the presentinvention may comprise a polyA-G Quartet. The nucleic acids and/or mRNAwith a polyA-G Quartet may further comprise a 5′ cap structure.

In one embodiment, the chain terminating nucleoside which may be used tostabilize the nucleic acid or mRNA comprising a polyA tail or polyA-GQuartet may be, but is not limited to, those described in InternationalPatent Publication No. WO2013103659, herein incorporated by reference inits entirety. In another embodiment, the chain terminating nucleosideswhich may be used with the present invention includes, but is notlimited to, 3′-deoxyadenosine (cordycepin), 3′-deoxyuridine,3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine,2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine,2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine,2′,3′-dideoxythymine, a 2′-deoxynucleoside, or a —O— methylnucleoside.

In another embodiment, the nucleic acid such as, but not limited tomRNA, which comprise a polyA tail or a polyA-G Quartet may be stabilizedby a modification to the 3′ region of the nucleic acid that can preventand/or inhibit the addition of oligo(U) (see e.g., International PatentPublication No. WO2013103659, herein incorporated by reference in itsentirety).

In yet another embodiment, the nucleic acid such as, but not limited tomRNA, which comprise a polyA tail or a polyA-G Quartet may be stabilizedby the addition of an oligonucleotide that terminates in a3′-deoxynucleoside, 2′,3′-dideoxynucleoside 3′-0-methylnucleosides,3′-0-ethylnucleosides, 3′-arabinosides, and other modified nucleosidesknown in the art and/or described herein.

Quantification

In one embodiment, the polynucleotides, primary constructs, modifiednucleic acids or mmRNA of the present invention may be quantified inexosomes derived from one or more bodily fluid. As used herein “bodilyfluids” include peripheral blood, serum, plasma, ascites, urine,cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid,aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolarlavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatoryfluid, sweat, fecal matter, hair, tears, cyst fluid, pleural andperitoneal fluid, pericardial fluid, lymph, chyme, chyle, bile,interstitial fluid, menses, pus, sebum, vomit, vaginal secretions,mucosal secretion, stool water, pancreatic juice, lavage fluids fromsinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, andumbilical cord blood. Alternatively, exosomes may be retrieved from anorgan selected from the group consisting of lung, heart, pancreas,stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast,prostate, brain, esophagus, liver, and placenta.

In the quantification method, a sample of not more than 2 mL is obtainedfrom the subject and the exosomes isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.In the analysis, the level or concentration of the polynucleotides,primary construct, modified nucleic acid or mmRNA may be an expressionlevel, presence, absence, truncation or alteration of the administeredconstruct. It is advantageous to correlate the level with one or moreclinical phenotypes or with an assay for a human disease biomarker. Theassay may be performed using construct specific probes, cytometry,qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, massspectrometry, or combinations thereof while the exosomes may be isolatedusing immunohistochemical methods such as enzyme linked immunosorbentassay (ELISA) methods. Exosomes may also be isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of the polynucleotides, primary constructs, modifiednucleic acid or mmRNA remaining or delivered. This is possible becausethe polynucleotides, primary constructs, modified nucleic acid or mmRNAof the present invention differ from the endogenous forms due to thestructural and/or chemical modifications.

II. Design and Synthesis of Polynucleotides

Polynucleotides, primary constructs modified nucleic acids or mmRNA foruse in accordance with the invention may be prepared according to anyavailable technique including, but not limited to chemical synthesis,enzymatic synthesis, which is generally termed in vitro transcription(IVT) or enzymatic or chemical cleavage of a longer precursor, etc.Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M.J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford[Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P.(ed.) Oligonucleotide synthesis: methods and applications, Methods inMolecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press,2005; both of which are incorporated herein by reference).

The process of design and synthesis of the primary constructs of theinvention generally includes the steps of gene construction, mRNAproduction (either with or without modifications) and purification. Inthe enzymatic synthesis method, a target polynucleotide sequenceencoding the polypeptide of interest is first selected for incorporationinto a vector which will be amplified to produce a cDNA template.Optionally, the target polynucleotide sequence and/or any flankingsequences may be codon optimized. The cDNA template is then used toproduce mRNA through in vitro transcription (IVT). After production, themRNA may undergo purification and clean-up processes. The steps of whichare provided in more detail below.

Gene Construction

The step of gene construction may include, but is not limited to genesynthesis, vector amplification, plasmid purification, plasmidlinearization and clean-up, and cDNA template synthesis and clean-up.

Gene Synthesis

Once a polypeptide of interest, or target, is selected for production, aprimary construct is designed. Within the primary construct, a firstregion of linked nucleosides encoding the polypeptide of interest may beconstructed using an open reading frame (ORF) of a selected nucleic acid(DNA or RNA) transcript. The ORF may comprise the wild type ORF, anisoform, variant or a fragment thereof. As used herein, an “open readingframe” or “ORF” is meant to refer to a nucleic acid sequence (DNA orRNA) which is capable of encoding a polypeptide of interest. ORFs oftenbegin with the start codon, ATG and end with a nonsense or terminationcodon or signal.

Further, the nucleotide sequence of the first region may be codonoptimized. Codon optimization methods are known in the art and may beuseful in efforts to achieve one or more of several goals. These goalsinclude to match codon frequencies in target and host organisms toensure proper folding, bias GC content to increase mRNA stability orreduce secondary structures, minimize tandem repeat codons or base runsthat may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeprotein trafficking sequences, remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, to adjusttranslational rates to allow the various domains of the protein to foldproperly, or to reduce or eliminate problem secondary structures withinthe mRNA. Codon optimization tools, algorithms and services are known inthe art, non-limiting examples include services from GeneArt (LifeTechnologies) and/or DNA2.0 (Menlo Park Calif.). In one embodiment, theORF sequence is optimized using optimization algorithms. Codon optionsfor each amino acid are given in Table 5.

TABLE 5 Codon Options Single Amino Acid Letter Code Codon OptionsIsoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA, CTG, TTA, TTG ValineV GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC Methionine M ATG CysteineC TGT, TGC Alanine A GCT, GCC, GCA, GCG Glycine G GGT, GGC, GGA, GGGProline P CCT, CCC, CCA, CCG Threonine T ACT, ACC, ACA, ACG Serine STCT, TCC, TCA, TCG, AGT, AGC Tyrosine Y TAT, TAC Tryptophan W TGGGlutamine Q CAA, CAG Asparagine N AAT, AAC Histidine H CAT, CAC Glutamicacid E GAA, GAG Aspartic acid D GAT, GAC Lysine K AAA, AAG Arginine RCGT, CGC, CGA, CGG, AGA, AGG Selenocysteine Sec UGA in mRNA in presenceof Selenocystein insertion element (SECIS) Stop codons Stop TAA, TAG,TGA

In one embodiment, after a nucleotide sequence has been codon optimizedit may be further evaluated for regions containing restriction sites. Atleast one nucleotide within the restriction site regions may be replacedwith another nucleotide in order to remove the restriction site from thesequence but the replacement of nucleotides does alter the amino acidsequence which is encoded by the codon optimized nucleotide sequence.

Features, which may be considered beneficial in some embodiments of thepresent invention, may be encoded by the primary construct and may flankthe ORF as a first or second flanking region. The flanking regions maybe incorporated into the primary construct before and/or afteroptimization of the ORF. It is not required that a primary constructcontain both a 5′ and 3′ flanking region. Examples of such featuresinclude, but are not limited to, untranslated regions (UTRs), Kozaksequences, an oligo(dT) sequence, and detectable tags and may includemultiple cloning sites which may have XbaI recognition.

In some embodiments, a 5′ UTR and/or a 3′ UTR may be provided asflanking regions. Multiple 5′ or 3′ UTRs may be included in the flankingregions and may be the same or of different sequences. Any portion ofthe flanking regions, including none, may be codon optimized and any mayindependently contain one or more different structural or chemicalmodifications, before and/or after codon optimization. Combinations offeatures may be included in the first and second flanking regions andmay be contained within other features. For example, the ORF may beflanked by a 5′ UTR which may contain a strong Kozak translationalinitiation signal and/or a 3′ UTR which may include an oligo(dT)sequence for templated addition of a poly-A tail.

Tables 2 and 3 provide a listing of exemplary UTRs which may be utilizedin the primary construct of the present invention as flanking regions.Shown in Table 6 is a representative listing of a 5′-untranslated regionof the invention. Variants of 5′ UTRs may be utilized wherein one ormore nucleotides are added or removed to the termini, including A, T, Cor G.

TABLE 6 5′-Untranslated Regions 5′ UTR Name/ SEQ ID IdentifierDescription Sequence NO. Native Wild type See wild type sequence — UTR5UTR-001 Synthetic GGGAAATAAGAGAGAAAAGAAGAGTAAG 5 UTRAAGAAATATAAGAGCCACC 5UTR-002 Upstream GGGAGATCAGAGAGAAAAGAAGAGTAAGA 6UTR AGAAATATAAGAGCCACC 5UTR-003 Upstream GGAATAAAAGTCTCAACACAACATATACA 7UTR AAACAAACGAATCTCAAGCAATCAAGCAT TCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAAT TTTCACCATTTACGAACGATAGCAAC 5UTR-004Upstream GGGAGACAAGCUUGGCAUUCCGGUACUGU 8 UTR UGGUAAAGCCACC

In another embodiment, the 5′ UTR may comprise a first polynucleotidefragment and a second polynucleotide fragment where the first and secondfragments may be from the same or different gene. (See e.g.,US20100293625 and US20110247090, each of which is herein incorporated byreference in its entirety). As a non-limiting example, the firstpolynucleotide may be a fragment of the canine, human or mouse SERCA2gene and/or the second polynucleotide fragment is a fragment of thebovine, mouse, rat or sheep beta-casein gene.

In one embodiment, the first polynucleotide fragment may be located onthe 5′ end of the second polynucleotide fragment. (See e.g.,US20100293625 and US20110247090, each of which is herein incorporated byreference in its entirety).

In another embodiment, the first polynucleotide fragment may comprisethe second intron of a sarcoplasmic/endoplasmic reticulum calcium ATPasegene and/or the second polynucleotide fragment comprises at least aportion of the 5′ UTR of a eukaryotic casein gene. (See e.g.,US20100293625 and US20110247090, each of which is herein incorporated byreference in its entirety). The first polynucleotide fragment may alsocomprise at least a portion of exon 2 and/or exon 3 of thesarcoplasmic/endoplasmic reticulum calcium ATPase gene. (See e.g.,US20100293625 and US20110247090, each of which is herein incorporated byreference in its entirety).

Shown in Table 7 is a representative listing of 3′-untranslated regionsof the invention. Variants of 3′ UTRs may be utilized wherein one ormore nucleotides are added or removed to the termini, including A, T, Cor G.

TABLE 7 3′-Untranslated Regions SEQ 3′ UTR Name/ ID IdentifierDescription Sequence NO. 3UTR-001 CreatineGCGCCTGCCCACCTGCCACCGACTGCTGGAAC 9 KinaseCCAGCCAGTGGGAGGGCCTGGCCCACCAGAGT CCTGCTCCCTCACTCCTCGCCCCGCCCCCTGTCCCAGAGTCCCACCTGGGGGCTCTCTCCACCCTT CTCAGAGTTCCAGTTTCAACCAGAGTTCCAACCAATGGGCTCCATCCTCTGGATTCTGGCCAATGA AATATCTCCCTGGCAGGGTCCTCTTCTTTTCCCAGAGCTCCACCCCAACCAGGAGCTCTAGTTAA TGGAGAGCTCCCAGCACACTCGGAGCTTGTGCTTTGTCTCCACGCAAAGCGATAAATAAAAGCA TTGGTGGCCTTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-002 Myoglobin GCCCCTGCCGCTCCCACCCCCACCCATCTGGGC 10CCCGGGTTCAAGAGAGAGCGGGGTCTGATCTC GTGTAGCCATATAGAGTTTGCTTCTGAGTGTCTGCTTTGTTTAGTAGAGGTGGGCAGGAGGAGCT GAGGGGCTGGGGCTGGGGTGTTGAAGTTGGCTTTGCATGCCCAGCGATGCGCCTCCCTGTGGGAT GTCATCACCCTGGGAACCGGGAGTGGCCCTTGGCTCACTGTGTTCTGCATGGTTTGGATCTGAAT TAATTGTCCTTTCTTCTAAATCCCAACCGAACTTCTTCCAACCTCCAAACTGGCTGTAACCCCAAA TCCAAGCCATTAACTACACCTGACAGTAGCAATTGTCTGATTAATCACTGGCCCCTTGAAGACAG CAGAATGTCCCTTTGCAATGAGGAGGAGATCTGGGCTGGGCGGGCCAGCTGGGGAAGCATTTGA CTATCTGGAACTTGTGTGTGCCTCCTCAGGTATGGCAGTGACTCACCTGGTTTTAATAAAACAAC CTGCAACATCTCATGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-003 α-actin ACACACTCCACCTCCAGCACGCGACTTCTCAG 11GACGACGAATCTTCTCAATGGGGGGGCGGCTG AGCTCCAGCCACCCCGCAGTCACTTTCTTTGTAACAACTTCCGTTGCTGCCATCGTAAACTGACAC AGTGTTTATAACGTGTACATACATTAACTTATTACCTCATTTTGTTATTTTTCGAAACAAAGCCCT GTGGAAGAAAATGGAAAACTTGAAGAAGCATTAAAGTCATTCTGTTAAGCTGCGTAAATGGTCTT TGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-004Albumin CATCACATTTAAAAGCATCTCAGCCTACCATG 12AGAATAAGAGAAAGAAAATGAAGATCAAAAG CTTATTCATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTC TTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAATCTAATAGAGTG GTACAGCACTGTTATTTTTCAAAGATGTGTTGCTATCCTGAAAATTCTGTAGGTTCTGTGGAAGTT CCAGTGTTCTCTCTTATTCCACTTCGGTAGAGGATTTCTAGTTTCTTGTGGGCTAATTAAATAAAT CATTAATACTCTTCTAATGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-005 α-globin GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATG13 CCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGG TCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 3UTR-006 G-CSF GCCAAGCCCTCCCCATCCCATGTATTTATCTCT 14ATTTAATATTTATGTCTATTTAAGCCTCATATTT AAAGACAGGGAAGAGCAGAACGGAGCCCCAGGCCTCTGTGTCCTTCCCTGCATTTCTGAGTTTC ATTCTCCTGCCTGTAGCAGTGAGAAAAAGCTCCTGTCCTCCCATCCCCTGGACTGGGAGGTAGAT AGGTAAATACCAAGTATTTATTACTATGACTGCTCCCCAGCCCTGGCTCTGCAATGGGCACTGGG ATGAGCCGCTGTGAGCCCCTGGTCCTGAGGGTCCCCACCTGGGACCCTTGAGAGTATCAGGTCT CCCACGTGGGAGACAAGAAATCCCTGTTTAATATTTAAACAGCAGTGTTCCCCATCTGGGTCCTT GCACCCCTCACTCTGGCCTCAGCCGACTGCACAGCGGCCCCTGCATCCCCTTGGCTGTGAGGCC CCTGGACAAGCAGAGGTGGCCAGAGCTGGGAGGCATGGCCCTGGGGTCCCACGAATTTGCTGG GGAATCTCGTTTTTCTTCTTAAGACTTTTGGGACATGGTTTGACTCCCGAACATCACCGACGCGT CTCCTGTTTTTCTGGGTGGCCTCGGGACACCTGCCCTGCCCCCACGAGGGTCAGGACTGTGACTC TTTTTAGGGCCAGGCAGGTGCCTGGACATTTGCCTTGCTGGACGGGGACTGGGGATGTGGGAGGG AGCAGACAGGAGGAATCATGTCAGGCCTGTGTGTGAAAGGAAGCTCCACTGTCACCCTCCACCT CTTCACCCCCCACTCACCAGTGTCCCCTCCACTGTCACATTGTAACTGAACTTCAGGATAATAAA GTGTTTGCCTCCATGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTA GA 3UTR-007 Col1a2;ACTCAATCTAAATTAAAAAAGAAAGAAATTTG 15 collagen,AAAAAACTTTCTCTTTGCCATTTCTTCTTCTTCT type I,TTTTTAACTGAAAGCTGAATCCTTCCATTTCTT alpha 2CTGCACATCTACTTGCTTAAATTGTGGGCAAAA GAGAAAAAGAAGGATTGATCAGAGCATTGTGCAATACAGTTTCATTAACTCCTTCCCCCGCTCCC CCAAAAATTTGAATTTTTTTTTCAACACTCTTACACCTGTTATGGAAAATGTCAACCTTTGTAAG AAAACCAAAATAAAAATTGAAAAATAAAAACCATAAACATTTGCACCACTTGTGGCTTTTGAAT ATCTTCCACAGAGGGAAGTTTAAAACCCAAACTTCCAAAGGTTTAAACTACCTCAAAACACTTTC CCATGAGTGTGATCCACATTGTTAGGTGCTGACCTAGACAGAGATGAACTGAGGTCCTTGTTTTGT TTTGTTCATAATACAAAGGTGCTAATTAATAGTATTTCAGATACTTGAAGAATGTTGATGGTGCTA GAAGAATTTGAGAAGAAATACTCCTGTATTGAGTTGTATCGTGTGGTGTATTTTTTAAAAAATTT GATTTAGCATTCATATTTTCCATCTTATTCCCAATTAAAAGTATGCAGATTATTTGCCCAAATCTT CTTCAGATTCAGCATTTGTTCTTTGCCAGTCTCATTTTCATCTTCTTCCATGGTTCCACAGAAGCT TTGTTTCTTGGGCAAGCAGAAAAATTAAATTGTACCTATTTTGTATATGTGAGATGTTTAAATAAA TTGTGAAAAAAATGAAATAAAGCATGTTTGGTTTTCCAAAAGAACATAT 3UTR-008 Col6a2; CGCCGCCGCCCGGGCCCCGCAGTCGAGGGTCG 16collagen, TGAGCCCACCCCGTCCATGGTGCTAAGCGGGC type VI,CCGGGTCCCACACGGCCAGCACCGCTGCTCAC alpha 2TCGGACGACGCCCTGGGCCTGCACCTCTCCAG CTCCTCCCACGGGGTCCCCGTAGCCCCGGCCCCCGCCCAGCCCCAGGTCTCCCCAGGCCCTCCG CAGGCTGCCCGGCCTCCCTCCCCCTGCAGCCATCCCAAGGCTCCTGACCTACCTGGCCCCTGAGCT CTGGAGCAAGCCCTGACCCAATAAAGGCTTTGAACCCAT 3UTR-009 RPN1; GGGGCTAGAGCCCTCTCCGCACAGCGTGGAGA 17 ribophorin ICGGGGCAAGGAGGGGGGTTATTAGGATTGGTG GTTTTGTTTTGCTTTGTTTAAAGCCGTGGGAAAATGGCACAACTTTACCTCTGTGGGAGATGCAA CACTGAGAGCCAAGGGGTGGGAGTTGGGATAATTTTTATATAAAAGAAGTTTTTCCACTTTGAAT TGCTAAAAGTGGCATTTTTCCTATGTGCAGTCACTCCTCTCATTTCTAAAATAGGGACGTGGCCAG GCACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGCTCACGAGGTCA GGAGATCGAGACTATCCTGGCTAACACGGTAAAACCCTGTCTCTACTAAAAGTACAAAAAATTA GCTGGGCGTGGTGGTGGGCACCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAAAGGC ATGAATCCAAGAGGCAGAGCTTGCAGTGAGCTGAGATCACGCCATTGCACTCCAGCCTGGGCAA CAGTGTTAAGACTCTGTCTCAAATATAAATAAATAAATAAATAAATAAATAAATAAATAAAAAT AAAGCGAGATGTTGCCCTCAAA 3UTR-010 LRP1;low GGCCCTGCCCCGTCGGACTGCCCCCAGAAAGC 18 densityCTCCTGCCCCCTGCCAGTGAAGTCCTTCAGTGA lipoproteinGCCCCTCCCCAGCCAGCCCTTCCCTGGCCCCGC receptor-CGGATGTATAAATGTAAAAATGAAGGAATTAC relatedATTTTATATGTGAGCGAGCAAGCCGGCAAGCG protein 1AGCACAGTATTATTTCTCCATCCCCTCCCTGCC TGCTCCTTGGCACCCCCATGCTGCCTTCAGGGAGACAGGCAGGGAGGGCTTGGGGCTGCACCTCC TACCCTCCCACCAGAACGCACCCCACTGGGAGAGCTGGTGGTGCAGCCTTCCCCTCCCTGTATAA GACACTTTGCCAAGGCTCTCCCCTCTCGCCCCATCCCTGCTTGCCCGCTCCCACAGCTTCCTGAGG GCTAATTCTGGGAAGGGAGAGTTCTTTGCTGCCCCTGTCTGGAAGACGTGGCTCTGGGTGAGGT AGGCGGGAAAGGATGGAGTGTTTTAGTTCTTGGGGGAGGCCACCCCAAACCCCAGCCCCAACTC CAGGGGCACCTATGAGATGGCCATGCTCAACCCCCCTCCCAGACAGGCCCTCCCTGTCTCCAGG GCCCCCACCGAGGTTCCCAGGGCTGGAGACTTCCTCTGGTAAACATTCCTCCAGCCTCCCCTCCC CTGGGGACGCCAAGGAGGTGGGCCACACCCAGGAAGGGAAAGCGGGCAGCCCCGTTTTGGGGAC GTGAACGTTTTAATAATTTTTGCTGAATTCCTTTACAACTAAATAACACAGATATTGTTATAAAT AAAATTGT 3UTR-011 Nnt1;ATATTAAGGATCAAGCTGTTAGCTAATAATGC 19 cardiotrophin-CACCTCTGCAGTTTTGGGAACAGGCAAATAAA like GTATCAGTATACATGGTGATGTACATCTGTAGCcytokine AAAGCTCTTGGAGAAAATGAAGACTGAAGAA factor 1AGCAAAGCAAAAACTGTATAGAGAGATTTTTC AAAAGCAGTAATCCCTCAATTTTAAAAAAGGATTGAAAATTCTAAATGTCTTTCTGTGCATATTT TTTGTGTTAGGAATCAAAAGTATTTTATAAAAGGAGAAAGAACAGCCTCATTTTAGATGTAGTCC TGTTGGATTTTTTATGCCTCCTCAGTAACCAGAAATGTTTTAAAAAACTAAGTGTTTAGGATTTCA AGACAACATTATACATGGCTCTGAAATATCTGACACAATGTAAACATTGCAGGCACCTGCATTT TATGTTTTTTTTTTCAACAAATGTGACTAATTTGAAACTTTTATGAACTTCTGAGCTGTCCCCTTG CAATTCAACCGCAGTTTGAATTAATCATATCAAATCAGTTTTAATTTTTTAAATTGTACTTCAGAG TCTATATTTCAAGGGCACATTTTCTCACTACTATTTTAATACATTAAAGGACTAAATAATCTTTCA GAGATGCTGGAAACAAATCATTTGCTTTATATGTTTCATTAGAATACCAATGAAACATACAACT TGAAAATTAGTAATAGTATTTTTGAAGATCCCATTTCTAATTGGAGATCTCTTTAATTTCGATCAA CTTATAATGTGTAGTACTATATTAAGTGCACTTGAGTGGAATTCAACATTTGACTAATAAAATGA GTTCATCATGTTGGCAAGTGATGTGGCAATTATCTCTGGTGACAAAAGAGTAAAATCAAATATTT CTGCCTGTTACAAATATCAAGGAAGACCTGCTACTATGAAATAGATGACATTAATCTGTCTTCAC TGTTTATAATACGGATGGATTTTTTTTCAAATCAGTGTGTGTTTTGAGGTCTTATGTAATTGATGA CATTTGAGAGAAATGGTGGCTTTTTTTAGCTACCTCTTTGTTCATTTAAGCACCAGTAAAGATCAT GTCTTTTTATAGAAGTGTAGATTTTCTTTGTGACTTTGCTATCGTGCCTAAAGCTCTAAATATAGG TGAATGTGTGATGAATACTCAGATTATTTGTCTCTCTATATAATTAGTTTGGTACTAAGTTTCTCA AAAAATTATTAACACATGAAAGACAATCTCTAAACCAGAAAAAGAAGTAGTACAAATTTTGTTA CTGTAATGCTCGCGTTTAGTGAGTTTAAAACACACAGTATCTTTTGGTTTTATAATCAGTTTCTATT TTGCTGTGCCTGAGATTAAGATCTGTGTATGTGTGTGTGTGTGTGTGTGCGTTTGTGTGTTAAAGC AGAAAAGACTTTTTTAAAAGTTTTAAGTGATAAATGCAATTTGTTAATTGATCTTAGATCACTAG TAAACTCAGGGCTGAATTATACCATGTATATTCTATTAGAAGAAAGTAAACACCATCTTTATTCCT GCCCTTTTTCTTCTCTCAAAGTAGTTGTAGTTATATCTAGAAAGAAGCAATTTTGATTTCTTGAAA AGGTAGTTCCTGCACTCAGTTTAAACTAAAAATAATCATACTTGGATTTTATTTATTTTTGTCATA GTAAAAATTTTAATTTATATATATTTTTATTTAGTATTATCTTATTCTTTGCTATTTGCCAATCCTT TGTCATCAATTGTGTTAAATGAATTGAAAATTCATGCCCTGTTCATTTTATTTTACTTTATTGGTTA GGATATTTAAAGGATTTTTGTATATATAATTTCTTAAATTAATATTCCAAAAGGTTAGTGGACTTA GATTATAAATTATGGCAAAAATCTAAAAACAACAAAAATGATTTTTATACATTCTATTTCATTAT TCCTCTTTTTCCAATAAGTCATACAATTGGTAGATATGACTTATTTTATTTTTGTATTATTCACTAT ATCTTTATGATATTTAAGTATAAATAATTAAAAAAATTTATTGTACCTTATAGTCTGTCACCAAAA AAAAAAAATTATCTGTAGGTAGTGAAATGCTAATGTTGATTTGTCTTTAAGGGCTTGTTAACTAT CCTTTATTTTCTCATTTGTCTTAAATTAGGAGTTTGTGTTTAAATTACTCATCTAAGCAAAAAATGT ATATAAATCCCATTACTGGGTATATACCCAAAGGATTATAAATCATGCTGCTATAAAGACACAT GCACACGTATGTTTATTGCAGCACTATTCACAATAGCAAAGACTTGGAACCAACCCAAATGTCCA TCAATGATAGACTTGATTAAGAAAATGTGCACATATACACCATGGAATACTATGCAGCCATAAA AAAGGATGAGTTCATGTCCTTTGTAGGGACATGGATAAAGCTGGAAACCATCATTCTGAGCAAA CTATTGCAAGGACAGAAAACCAAACACTGCATGTTCTCACTCATAGGTGGGAATTGAACAATGA GAACACTTGGACACAAGGTGGGGAACACCACACACCAGGGCCTGTCATGGGGTGGGGGGAGTGG GGAGGGATAGCATTAGGAGATATACCTAATGTAAATGATGAGTTAATGGGTGCAGCACACCAAC ATGGCACATGTATACATATGTAGCAAACCTGCACGTTGTGCACATGTACCCTAGAACTTAAAGT ATAATTAAAAAAAAAAAGAAAACAGAAGCTATTTATAAAGAAGTTATTTGCTGAAATAAATGTG ATCTTTCCCATTAAAAAAATAAAGAAATTTTGGGGTAAAAAAACACAATATATTGTATTCTTGA AAAATTCTAAGAGAGTGGATGTGAAGTGTTCTCACCACAAAAGTGATAACTAATTGAGGTAATG CACATATTAATTAGAAAGATTTTGTCATTCCACAATGTATATATACTTAAAAATATGTTATACACA ATAAATACATACATTAAAAAATAAGTAAATGTA3UTR-012 Col6a1; CCCACCCTGCACGCCGGCACCAAACCCTGTCC 20 collagen,TCCCACCCCTCCCCACTCATCACTAAACAGAGT type VI,AAAATGTGATGCGAATTTTCCCGACCAACCTG alpha 1ATTCGCTAGATTTTTTTTAAGGAAAAGCTTGGA AAGCCAGGACACAACGCTGCTGCCTGCTTTGTGCAGGGTCCTCCGGGGCTCAGCCCTGAGTTGG CATCACCTGCGCAGGGCCCTCTGGGGCTCAGCCCTGAGCTAGTGTCACCTGCACAGGGCCCTCT GAGGCTCAGCCCTGAGCTGGCGTCACCTGTGCAGGGCCCTCTGGGGCTCAGCCCTGAGCTGGCC TCACCTGGGTTCCCCACCCCGGGCTCTCCTGCCCTGCCCTCCTGCCCGCCCTCCCTCCTGCCTGCG CAGCTCCTTCCCTAGGCACCTCTGTGCTGCATCCCACCAGCCTGAGCAAGACGCCCTCTCGGGGC CTGTGCCGCACTAGCCTCCCTCTCCTCTGTCCCCATAGCTGGTTTTTCCCACCAATCCTCACCTAA CAGTTACTTTACAATTAAACTCAAAGCAAGCTCTTCTCCTCAGCTTGGGGCAGCCATTGGCCTCT GTCTCGTTTTGGGAAACCAAGGTCAGGAGGCCGTTGCAGACATAAATCTCGGCGACTCGGCCCC GTCTCCTGAGGGTCCTGCTGGTGACCGGCCTGGACCTTGGCCCTACAGCCCTGGAGGCCGCTGC TGACCAGCACTGACCCCGACCTCAGAGAGTACTCGCAGGGGCGCTGGCTGCACTCAAGACCCTC GAGATTAACGGTGCTAACCCCGTCTGCTCCTCCCTCCCGCAGAGACTGGGGCCTGGACTGGACAT GAGAGCCCCTTGGTGCCACAGAGGGCTGTGTCTTACTAGAAACAACGCAAACCTCTCCTTCCTCA GAATAGTGATGTGTTCGACGTTTTATCAAAGGCCCCCTTTCTATGTTCATGTTAGTTTTGCTCCTT CTGTGTTTTTTTCTGAACCATATCCATGTTGCTGACTTTTCCAAATAAAGGTTTTCACTCCTCTC 3UTR-013 Calr;AGAGGCCTGCCTCCAGGGCTGGACTGAGGCCT 21 calreticulinGAGCGCTCCTGCCGCAGAGCTGGCCGCGCCAA ATAATGTCTCTGTGAGACTCGAGAACTTTCATTTTTTTCCAGGCTGGTTCGGATTTGGGGTGGATT TTGGTTTTGTTCCCCTCCTCCACTCTCCCCCACCCCCTCCCCGCCCTTTTTTTTTTTTTTTTTTAAAC TGGTATTTTATCTTTGATTCTCCTTCAGCCCTCACCCCTGGTTCTCATCTTTCTTGATCAACATCTTT TCTTGCCTCTGTCCCCTTCTCTCATCTCTTAGCTCCCCTCCAACCTGGGGGGCAGTGGTGTGGAGA AGCCACAGGCCTGAGATTTCATCTGCTCTCCTTCCTGGAGCCCAGAGGAGGGCAGCAGAAGGGG GTGGTGTCTCCAACCCCCCAGCACTGAGGAAGAACGGGGCTCTTCTCATTTCACCCCTCCCTTTC TCCCCTGCCCCCAGGACTGGGCCACTTCTGGGTGGGGCAGTGGGTCCCAGATTGGCTCACACTGA GAATGTAAGAACTACAAACAAAATTTCTATTAAATTAAATTTTGTGTCTCC 3UTR-014 Colla1; CTCCCTCCATCCCAACCTGGCTCCCTCCCACCC22 collagen, AACCAACTTTCCCCCCAACCCGGAAACAGACA type I,AGCAACCCAAACTGAACCCCCTCAAAAGCCAA alpha 1AAAATGGGAGACAATTTCACATGGACTTTGGA AAATATTTTTTTCCTTTGCATTCATCTCTCAAACTTAGTTTTTATCTTTGACCAACCGAACATGACC AAAAACCAAAAGTGCATTCAACCTTACCAAAAAAAAAAAAAAAAAAAGAATAAATAAATAACT TTTTAAAAAAGGAAGCTTGGTCCACTTGCTTGAAGACCCATGCGGGGGTAAGTCCCTTTCTGCCC GTTGGGCTTATGAAACCCCAATGCTGCCCTTTCTGCTCCTTTCTCCACACCCCCCTTGGGGCCTCC CCTCCACTCCTTCCCAAATCTGTCTCCCCAGAAGACACAGGAAACAATGTATTGTCTGCCCAGCA ATCAAAGGCAATGCTCAAACACCCAAGTGGCCCCCACCCTCAGCCCGCTCCTGCCCGCCCAGCA CCCCCAGGCCCTGGGGGACCTGGGGTTCTCAGACTGCCAAAGAAGCCTTGCCATCTGGCGCTCC CATGGCTCTTGCAACATCTCCCCTTCGTTTTTGAGGGGGTCATGCCGGGGGAGCCACCAGCCCCT CACTGGGTTCGGAGGAGAGTCAGGAAGGGCCACGACAAAGCAGAAACATCGGATTTGGGGAACG CGTGTCAATCCCTTGTGCCGCAGGGCTGGGCGGGAGAGACTGTTCTGTTCCTTGTGTAACTGTGT TGCTGAAAGACTACCTCGTTCTTGTCTTGATGTGTCACCGGGGCAACTGCCTGGGGGCGGGGATG GGGGCAGGGTGGAAGCGGCTCCCCATTTTATACCAAAGGTGCTACATCTATGTGATGGGTGGGG TGGGGAGGGAATCACTGGTGCTATAGAAATTGAGATGCCCCCCCAGGCCAGCAAATGTTCCTTTT TGTTCAAAGTCTATTTTTATTCCTTGATATTTTTCTTTTTTTTTTTTTTTTTTTGTGGATGGGGACTT GTGAATTTTTCTAAAGGTGCTATTTAACATGGGAGGAGAGCGTGTGCGGCTCCAGCCCAGCCCGC TGCTCACTTTCCACCCTCTCTCCACCTGCCTCTGGCTTCTCAGGCCTCTGCTCTCCGACCTCTCTC CTCTGAAACCCTCCTCCACAGCTGCAGCCCATCCTCCCGGCTCCCTCCTAGTCTGTCCTGCGTCCT CTGTCCCCGGGTTTCAGAGACAACTTCCCAAAGCACAAAGCAGTTTTTCCCCCTAGGGGTGGGA GGAAGCAAAAGACTCTGTACCTATTTTGTATGTGTATAATAATTTGAGATGTTTTTAATTATTTTG ATTGCTGGAATAAAGCATGTGGAAATGACCCAAACATAATCCGCAGTGGCCTCCTAATTTCCTTC TTTGGAGTTGGGGGAGGGGTAGACATGGGGAAGGGGCTTTGGGGTGATGGGCTTGCCTTCCATTC CTGCCCTTTCCCTCCCCACTATTCTCTTCTAGATCCCTCCATAACCCCACTCCCCTTTCTCTCACCC TTCTTATACCGCAAACCTTTCTACTTCCTCTTTCATTTTCTATTCTTGCAATTTCCTTGCACCTTTTC CAAATCCTCTTCTCCCCTGCAATACCATACAGGCAATCCACGTGCACAACACACACACACACTCT TCACATCTGGGGTTGTCCAAACCTCATACCCACTCCCCTTCAAGCCCATCCACTCTCCACCCCCTG GATGCCCTGCACTTGGTGGCGGTGGGATGCTCATGGATACTGGGAGGGTGAGGGGAGTGGAAC CCGTGAGGAGGACCTGGGGGCCTCTCCTTGAACTGACATGAAGGGTCATCTGGCCTCTGCTCCCT TCTCACCCACGCTGACCTCCTGCCGAAGGAGCAACGCAACAGGAGAGGGGTCTGCTGAGCCTGG CGAGGGTCTGGGAGGGACCAGGAGGAAGGCGTGCTCCCTGCTCGCTGTCCTGGCCCTGGGGGAG TGAGGGAGACAGACACCTGGGAGAGCTGTGGGGAAGGCACTCGCACCGTGCTCTTGGGAAGGA AGGAGACCTGGCCCTGCTCACCACGGACTGGGTGCCTCGACCTCCTGAATCCCCAGAACACAAC CCCCCTGGGCTGGGGTGGTCTGGGGAACCATCGTGCCCCCGCCTCCCGCCTACTCCTTTTTAAGC TT 3UTR-015 Plod1;TTGGCCAGGCCTGACCCTCTTGGACCTTTCTTC 23 procollagen-TTTGCCGACAACCACTGCCCAGCAGCCTCTGG lysine, 2-GACCTCGGGGTCCCAGGGAACCCAGTCCAGCC oxoglutarateTCCTGGCTGTTGACTTCCCATTGCTCTTGGAGC 5- CACCAATCAAAGAGATTCAAAGAGATTCCTGCdioxygenase 1 AGGCCAGAGGCGGAACACACCTTTATGGCTGGGGCTCTCCGTGGTGTTCTGGACCCAGCCCCTGG AGACACCATTCACTTTTACTGCTTTGTAGTGACTCGTGCTCTCCAACCTGTCTTCCTGAAAAACCA AGGCCCCCTTCCCCCACCTCTTCCATGGGGTGAGACTTGAGCAGAACAGGGGCTTCCCCAAGTTG CCCAGAAAGACTGTCTGGGTGAGAAGCCATGGCCAGAGCTTCTCCCAGGCACAGGTGTTGCACC AGGGACTTCTGCTTCAAGTTTTGGGGTAAAGACACCTGGATCAGACTCCAAGGGCTGCCCTGAG TCTGGGACTTCTGCCTCCATGGCTGGTCATGAGAGCAAACCGTAGTCCCCTGGAGACAGCGACTC CAGAGAACCTCTTGGGAGACAGAAGAGGCATCTGTGCACAGCTCGATCTTCTACTTGCCTGTGGG GAGGGGAGTGACAGGTCCACACACCACACTGGGTCACCCTGTCCTGGATGCCTCTGAAGAGAGG GACAGACCGTCAGAAACTGGAGAGTTTCTATTAAAGGTCATTTAAACCA 3UTR-016 Nucb1; TCCTCCGGGACCCCAGCCCTCAGGATTCCTGAT 24nucleobindin 1 GCTCCAAGGCGACTGATGGGCGCTGGATGAAGTGGCACAGTCAGCTTCCCTGGGGGCTGGTGTC ATGTTGGGCTCCTGGGGCGGGGGCACGGCCTGGCATTTCACGCATTGCTGCCACCCCAGGTCCAC CTGTCTCCACTTTCACAGCCTCCAAGTCTGTGGCTCTTCCCTTCTGTCCTCCGAGGGGCTTGCCTT CTCTCGTGTCCAGTGAGGTGCTCAGTGATCGGCTTAACTTAGAGAAGCCCGCCCCCTCCCCTTCTC CGTCTGTCCCAAGAGGGTCTGCTCTGAGCCTGCGTTCCTAGGTGGCTCGGCCTCAGCTGCCTGGGT TGTGGCCGCCCTAGCATCCTGTATGCCCACAGCTACTGGAATCCCCGCTGCTGCTCCGGGCCAAG CTTCTGGTTGATTAATGAGGGCATGGGGTGGTCCCTCAAGACCTTCCCCTACCTTTTGTGGAACC AGTGATGCCTCAAAGACAGTGTCCCCTCCACAGCTGGGTGCCAGGGGCAGGGGATCCTCAGTAT AGCCGGTGAACCCTGATACCAGGAGCCTGGGCCTCCCTGAACCCCTGGCTTCCAGCCATCTCATC GCCAGCCTCCTCCTGGACCTCTTGGCCCCCAGCCCCTTCCCCACACAGCCCCAGAAGGGTCCCAG AGCTGACCCCACTCCAGGACCTAGGCCCAGCCCCTCAGCCTCATCTGGAGCCCCTGAAGACCAG TCCCACCCACCTTTCTGGCCTCATCTGACACTGCTCCGCATCCTGCTGTGTGTCCTGTTCCATGTT CCGGTTCCATCCAAATACACTTTCTGGAACAAA3UTR-017 α-globin GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCT 25TGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTG CACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC

It should be understood that those listed in the previous tables areexamples and that any UTR from any gene may be incorporated into therespective first or second flanking region of the primary construct.Furthermore, multiple wild-type UTRs of any known gene may be utilized.It is also within the scope of the present invention to provideartificial UTRs which are not variants of wild type genes. These UTRs orportions thereof may be placed in the same orientation as in thetranscript from which they were selected or may be altered inorientation or location. Hence a 5′ or 3′ UTR may be inverted,shortened, lengthened, made chimeric with one or more other 5′ UTRs or3′ UTRs. As used herein, the term “altered” as it relates to a UTRsequence, means that the UTR has been changed in some way in relation toa reference sequence. For example, a 3′ or 5′ UTR may be alteredrelative to a wild type or native UTR by the change in orientation orlocation as taught above or may be altered by the inclusion ofadditional nucleotides, deletion of nucleotides, swapping ortransposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In one embodiment, a double, triple or quadruple UTR such as a 5′ or 3′UTR may be used. As used herein, a “double” UTR is one in which twocopies of the same UTR are encoded either in series or substantially inseries. For example, a double beta-globin 3′ UTR may be used asdescribed in US Patent publication 20100129877, the contents of whichare incorporated herein by reference in its entirety.

It is also within the scope of the present invention to have patternedUTRs. As used herein “patterned UTRs” are those UTRs which reflect arepeating or alternating pattern, such as ABABAB or AABBAABBAABB orABCABCABC or variants thereof repeated once, twice, or more than 3times. In these patterns, each letter, A, B, or C represent a differentUTR at the nucleotide level.

In one embodiment, flanking regions are selected from a family oftranscripts whose proteins share a common function, structure, featureof property. For example, polypeptides of interest may belong to afamily of proteins which are expressed in a particular cell, tissue orat some time during development. The UTRs from any of these genes may beswapped for any other UTR of the same or different family of proteins tocreate a new chimeric primary transcript. As used herein, a “family ofproteins” is used in the broadest sense to refer to a group of two ormore polypeptides of interest which share at least one function,structure, feature, localization, origin, or expression pattern.

After optimization (if desired), the primary construct components arereconstituted and transformed into a vector such as, but not limited to,plasmids, viruses, cosmids, and artificial chromosomes. For example, theoptimized construct may be reconstituted and transformed into chemicallycompetent E. coli, yeast, neurospora, maize, drosophila, etc. where highcopy plasmid-like or chromosome structures occur by methods describedherein. Stop Codons

In one embodiment, the primary constructs of the present invention mayinclude at least two stop codons before the 3′ untranslated region(UTR). The stop codon may be selected from TGA, TAA and TAG. In oneembodiment, the primary constructs of the present invention include thestop codon TGA and one additional stop codon. In a further embodimentthe addition stop codon may be TAA.

Vector Amplification

The vector containing the primary construct is then amplified and theplasmid isolated and purified using methods known in the art such as,but not limited to, a maxi prep using the Invitrogen PURELINK™ HiPureMaxiprep Kit (Carlsbad, Calif.).

Plasmid Linearization

The plasmid may then be linearized using methods known in the art suchas, but not limited to, the use of restriction enzymes and buffers. Thelinearization reaction may be purified using methods including, forexample Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.), andHPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen'sstandard PURELINK™ PCR Kit (Carlsbad, Calif.). The purification methodmay be modified depending on the size of the linearization reactionwhich was conducted. The linearized plasmid is then used to generatecDNA for in vitro transcription (IVT) reactions.

cDNA Template Synthesis

A cDNA template may be synthesized by having a linearized plasmidundergo polymerase chain reaction (PCR). Table 8 is a listing of primersand probes that may be useful in the PCR reactions of the presentinvention. It should be understood that the listing is not exhaustiveand that primer-probe design for any amplification is within the skillof those in the art. Probes may also contain chemically modified basesto increase base-pairing fidelity to the target molecule andbase-pairing strength. Such modifications may include 5-methyl-Cytidine,2,6-di-amino-purine, 2′-fluoro, phosphoro-thioate, or locked nucleicacids.

TABLE 8 Primers and Probes Primer/ Probe Hybridization SEQ ID IdentifierSequence (5′-3′) target NO. UFP TTGGACCCTCGTACAGAAGCTAA cDNA Template 26TACG URP T_(x160)CTTCCTACTCAGGCTTTATTC cDNA Template 27 AAAGACCA GBA1CCTTGACCTTCTGGAACTTC Acid 28 glucocerebrosidase GBA2CCAAGCACTGAAACGGATAT Acid 29 glucocerebrosidase LUC1GATGAAAAGTGCTCCAAGGA Luciferase 30 LUC2 AACCGTGATGAAAAGGTACC Luciferase31 LUC3 TCATGCAGATTGGAAAGGTC Luciferase 32 GCSF1 CTTCTTGGACTGTCCAGAGGG-CSF 33 GCSF2 GCAGTCCCTGATACAAGAAC G-CSF 34 GCSF3 GATTGAAGGTGGCTCGCTACG-CSF 35 *UFP is universal forward primer; URP is universal reverseprimer.

In one embodiment, the cDNA may be submitted for sequencing analysisbefore undergoing transcription.

Polynucleotide Production

The process of polynucleotide production may include, but is not limitedto, in vitro transcription, cDNA template removal and RNA clean-up, andcapping and/or tailing reactions.

In Vitro Transcription

The cDNA produced in the previous step may be transcribed using an invitro transcription (IVT) system. The system typically comprises atranscription buffer, nucleotide triphosphates (NTPs), an RNaseinhibitor and a polymerase. The NTPs may be manufactured in house, maybe selected from a supplier, or may be synthesized as described herein.The NTPs may be selected from, but are not limited to, those describedherein including natural and unnatural (modified) NTPs. The polymerasemay be selected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to be incorporated into modified nucleic acids.

RNA Polymerases

Any number of RNA polymerases or variants may be used in the design ofthe primary constructs of the present invention.

RNA polymerases may be modified by inserting or deleting amino acids ofthe RNA polymerase sequence. As a non-limiting example, the RNApolymerase may be modified to exhibit an increased ability toincorporate a 2′-modified nucleotide triphosphate compared to anunmodified RNA polymerase (see International Publication WO2008078180and U.S. Pat. No. 8,101,385; herein incorporated by reference in theirentireties).

Variants may be obtained by evolving an RNA polymerase, optimizing theRNA polymerase amino acid and/or nucleic acid sequence and/or by usingother methods known in the art. As a non-limiting example, T7 RNApolymerase variants may be evolved using the continuous directedevolution system set out by Esvelt et al. (Nature (2011)472(7344):499-503; herein incorporated by reference in its entirety)where clones of T7 RNA polymerase may encode at least one mutation suchas, but not limited to, lysine at position 93 substituted for threonine(K93T), 14M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T,N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A,Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P,A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A,H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E,N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limitingexample, T7 RNA polymerase variants may encode at least mutation asdescribed in U.S. Pub. Nos. 20100120024 and 20070117112; hereinincorporated by reference in their entireties. Variants of RNApolymerase may also include, but are not limited to, substitutionalvariants, conservative amino acid substitution, insertional variants,deletional variants and/or covalent derivatives.

In one embodiment, the primary construct may be designed to berecognized by the wild type or variant RNA polymerases. In doing so,primary construct may be modified to contain sites or regions ofsequence changes from the wild type or parent primary construct.

In one embodiment, the primary construct may be designed to include atleast one substitution and/or insertion upstream of an RNA polymerasebinding or recognition site, downstream of the RNA polymerase binding orrecognition site, upstream of the TATA box sequence, downstream of theTATA box sequence of the primary construct but upstream of the codingregion of the primary construct, within the 5′UTR, before the 5′UTRand/or after the 5′UTR.

In one embodiment, the 5′UTR of the primary construct may be replaced bythe insertion of at least one region and/or string of nucleotides of thesame base. The region and/or string of nucleotides may include, but isnot limited to, at least 3, at least 4, at least 5, at least 6, at least7 or at least 8 nucleotides and the nucleotides may be natural and/orunnatural. As a non-limiting example, the group of nucleotides mayinclude 5-8 adenine, cytosine, thymine, a string of any of the othernucleotides disclosed herein and/or combinations thereof.

In one embodiment, the 5′UTR of the primary construct may be replaced bythe insertion of at least two regions and/or strings of nucleotides oftwo different bases such as, but not limited to, adenine, cytosine,thymine, any of the other nucleotides disclosed herein and/orcombinations thereof. For example, the 5′UTR may be replaced byinserting 5-8 adenine bases followed by the insertion of 5-8 cytosinebases. In another example, the 5′UTR may be replaced by inserting 5-8cytosine bases followed by the insertion of 5-8 adenine bases.

In one embodiment, the primary construct may include at least onesubstitution and/or insertion downstream of the transcription start sitewhich may be recognized by an RNA polymerase. As a non-limiting example,at least one substitution and/or insertion may occur downstream thetranscription start site by substituting at least one nucleic acid inthe region just downstream of the transcription start site (such as, butnot limited to, +1 to +6). Changes to region of nucleotides justdownstream of the transcription start site may affect initiation rates,increase apparent nucleotide triphosphate (NTP) reaction constantvalues, and increase the dissociation of short transcripts from thetranscription complex curing initial transcription (Brieba et al,Biochemistry (2002) 41: 5144-5149; herein incorporated by reference inits entirety). The modification, substitution and/or insertion of atleast one nucleic acid may cause a silent mutation of the nucleic acidsequence or may cause a mutation in the amino acid sequence.

In one embodiment, the primary construct may include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12 or at least 13 guanine bases downstream of the transcription startsite.

In one embodiment, the primary construct may include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5 or at least 6guanine bases in the region just downstream of the transcription startsite. As a non-limiting example, if the nucleotides in the region areGGGAGA the guanine bases may be substituted by at least 1, at least 2,at least 3 or at least 4 adenine nucleotides. In another non-limitingexample, if the nucleotides in the region are GGGAGA the guanine basesmay be substituted by at least 1, at least 2, at least 3 or at least 4cytosine bases. In another non-limiting example, if the nucleotides inthe region are GGGAGA the guanine bases may be substituted by at least1, at least 2, at least 3 or at least 4 thymine, and/or any of thenucleotides described herein.

In one embodiment, the primary construct may include at least onesubstitution and/or insertion upstream of the start codon. For thepurpose of clarity, one of skill in the art would appreciate that thestart codon is the first codon of the protein coding region whereas thetranscription start site is the site where transcription begins. Theprimary construct may include, but is not limited to, at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7 orat least 8 substitutions and/or insertions of nucleotide bases. Thenucleotide bases may be inserted or substituted at 1, at least 1, atleast 2, at least 3, at least 4 or at least 5 locations upstream of thestart codon. The nucleotides inserted and/or substituted may be the samebase (e.g., all A or all C or all T or all G), two different bases(e.g., A and C, A and T, or C and T), three different bases (e.g., A, Cand T or A, C and T) or at least four different bases. As a non-limitingexample, the guanine base upstream of the coding region in the primaryconstruct may be substituted with adenine, cytosine, thymine, or any ofthe nucleotides described herein. In another non-limiting example thesubstitution of guanine bases in the primary construct may be designedso as to leave one guanine base in the region downstream of thetranscription start site and before the start codon (see Esvelt et al.Nature (2011) 472(7344):499-503; herein incorporated by reference in itsentirety). As a non-limiting example, at least 5 nucleotides may beinserted at 1 location downstream of the transcription start site butupstream of the start codon and the at least 5 nucleotides may be thesame base type.

cDNA Template Removal and Clean-Up

The cDNA template may be removed using methods known in the art such as,but not limited to, treatment with Deoxyribonuclease I (DNase I). RNAclean-up may also include a purification method such as, but not limitedto, AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, Mass.),HPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

Capping and/or Tailing Reactions

The primary construct or mmRNA may also undergo capping and/or tailingreactions. A capping reaction may be performed by methods known in theart to add a 5′ cap to the 5′ end of the primary construct. Methods forcapping include, but are not limited to, using a Vaccinia Capping enzyme(New England Biolabs, Ipswich, Mass.).

A poly-A tailing reaction may be performed by methods known in the art,such as, but not limited to, 2′ O-methyltransferase and by methods asdescribed herein. If the primary construct generated from cDNA does notinclude a poly-T, it may be beneficial to perform the poly-A-tailingreaction before the primary construct is cleaned.

Purification

The primary construct or mmRNA purification may include, but is notlimited to, mRNA or mmRNA clean-up, quality assurance and qualitycontrol. mRNA or mmRNA clean-up may be performed by methods known in thearts such as, but not limited to, AGENCOURT® beads (Beckman CoulterGenomics, Danvers, Mass.), poly-T beads, LNA™ oligo-T capture probes(EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods suchas, but not limited to, strong anion exchange HPLC, weak anion exchangeHPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC(HIC-HPLC). The term “purified” when used in relation to apolynucleotide such as a “purified mRNA or mmRNA” refers to one that isseparated from at least one contaminant. As used herein, a “contaminant”is any substance which makes another unfit, impure or inferior. Thus, apurified polynucleotide (e.g., DNA and RNA) is present in a form orsetting different from that in which it is found in nature, or a form orsetting different from that which existed prior to subjecting it to atreatment or purification method.

A quality assurance and/or quality control check may be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In another embodiment, the mRNA or mmRNA may be sequenced by methodsincluding, but not limited to reverse-transcriptase-PCR.

In one embodiment, the mRNA or mmRNA may be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, Mass.). The quantified mRNA ormmRNA may be analyzed in order to determine if the mRNA or mmRNA may beof proper size, check that no degradation of the mRNA or mmRNA hasoccurred. Degradation of the mRNA and/or mmRNA may be checked by methodssuch as, but not limited to, agarose gel electrophoresis, HPLC basedpurification methods such as, but not limited to, strong anion exchangeHPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), andhydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-massspectrometry (LCMS), capillary electrophoresis (CE) and capillary gelelectrophoresis (CGE).

Signal Peptides or Proteins

The primary constructs or mmRNA may also encode additional featureswhich facilitate trafficking of the polypeptides to therapeuticallyrelevant sites. One such feature which aids in protein trafficking isthe signal peptide sequence. As used herein, a “signal sequence” or“signal peptide” is a polynucleotide or polypeptide, respectively, whichis from about 9 to 200 nucleotides (3-60 amino acids) in length which isincorporated at the 5′ (or N-terminus) of the coding region orpolypeptide encoded, respectively. Addition of these sequences result intrafficking of the encoded polypeptide to the endoplasmic reticulumthrough one or more secretory pathways. Some signal peptides are cleavedfrom the protein by signal peptidase after the proteins are transported.

Table 9 is a representative listing of signal proteins or peptides whichmay be incorporated for encoding by the polynucleotides, primaryconstructs or mmRNA of the invention.

TABLE 9 Signal Peptides SEQ SEQ NUCLEOTIDE SEQUENCE ID ENCODED ID IDDescription (5′-3′) NO. PEPTIDE NO. SS- α-1- ATGATGCCATCCTCAGTCTCA 36MMPSSVSW 98 001 antitrypsin TGGGGTATTTTGCTCTTGGCG GILLAGLCCGGTCTGTGCTGTCTCGTGCCG LVPVSLA GTGTCGCTCGCA SS- G-CSFATGGCCGGACCGGCGACTCAG 37 MAGPATQS 99 002 TCGCCCATGAAACTCATGGCC PMKLMALQCTGCAGTTGTTGCTTTGGCAC LLLWHSAL TCAGCCCTCTGGACCGTCCAA WTVQEA GAGGCG SS-Factor IX ATGCAGAGAGTGAACATGATT 38 MQRVNMIM 100 003ATGGCCGAGTCCCCATCGCTC AESPSLITIC ATCACAATCTGCCTGCTTGGT LLGYLLSAEACCTGCTTTCCGCCGAATGCA CTVFLDHEN CTGTCTTTCTGGATCACGAGA ANKILNRPKRATGCGAATAAGATCTTGAACC GACCCAAACGG SS- Prolactin ATGAAAGGATCATTGCTGTTG 39MKGSLLLLL 101 004 CTCCTCGTGTCGAACCTTCTG VSNLLLCQS CTTTGCCAGTCCGTAGCCCCCVAP SS- Albumin ATGAAATGGGTGACGTTCATC 40 MKWVTFISL 102 005TCACTGTTGTTTTTGTTCTCGT LFLFSSAYS CCGCCTACTCCAGGGGAGTAT RG VFRR TCCGCCGASS- HMMSP38 ATGTGGTGGCGGCTCTGGTGG 41 MWWRLWW 103 006CTGCTCCTGTTGCTCCTCTTGC LLLLLLLLP TGTGGCCCATGGTGTGGGCA MWA MLS- ornithineTGCTCTTTAACCTCCGCATCCT 42 MLFNLRILL 104 001 carbamoyltransferaseGTTGAATAACGCTGCGTTCCG NNAAFRNG AAATGGGCATAACTTCATGGT HNFMVRNFACGCAACTTCAGATGCGGCCA RCGQPLQ GCCACTCCAG MLS- CytochromeATGTCCGTCTTGACACCCCTG 43 MSVLTPLLL 105 002 C OxidaseCTCTTGAGAGGGCTGACGGGG RGLTGSARR subunit 8A TCCGCTAGACGCCTGCCGGTALPVPRAKIH CCGCGAGCGAAGATCCACTCC SL CTG MLS- CytochromeATGAGCGTGCTCACTCCGTTG 44 MSVLTPLLL 106 003 C OxidaseCTTCTTCGAGGGCTTACGGGA RGLTGSARR subunit 8A TCGGCTCGGAGGTTGCCCGTCLPVPRAKIH CCGAGAGCGAAGATCCATTCG SL TTG SS- Type III,TGACAAAAATAACTTTATCTC 45 MVTKITLSP 107 007 bacterialCCCAGAATTTTAGAATCCAAA QNFRIQKQE AACAGGAAACCACACTACTA TTLLKEKSTAAAGAAAAATCAACCGAGAA EKNSLAKSI AAATTCTTTAGCAAAAAGTAT LAVKNHFIETCTCGCAGTAAAAATCACTTC LRSKLSERFI ATCGAATTAAGGTCAAAATTA SHKNTTCGGAACGTTTTATTTCGCAT AAGAACACT SS- Viral ATGCTGAGCTTTGTGGATACC 46MLSFVDTRT 108 008 CGCACCCTGCTGCTGCTGGCG LLLLAVTSC GTGACCAGCTGCCTGGCGACCLATCQ TGCCAG SS- viral ATGGGCAGCAGCCAGGCGCC 47 MGSSQAPR 109 009GCGCATGGGCAGCGTGGGCG MGSVGGHG GCCATGGCCTGATGGCGCTGC LMALLMAGTGATGGCGGGCCTGATTCTGC LILPGILA CGGGCATTCTGGCG SS- ViralATGGCGGGCATTTTTTATTTTC 48 MAGIFYFLF 110 010 TGTTTAGCTTTCTGTTTGGCATSFLFGICD TTGCGAT SS- Viral ATGGAAAACCGCCTGCTGCGC 49 MENRLLRV 111 011GTGTTTCTGGTGTGGGCGGCG FLVWAALT CTGACCATGGATGGCGCGAGC MDGASA GCG SS-Viral ATGGCGCGCCAGGGCTGCTTT 50 MARQGCFG 112 012 GGCAGCTATCAGGTGATTAGCSYQVISLFTF CTGTTTACCTTTGCGATTGGC AIGVNLCLG GTGAACCTGTGCCTGGGC SS-Bacillus ATGAGCCGCCTGCCGGTGCTG 51 MSRLPVLLL 113 013CTGCTGCTGCAGCTGCTGGTG LQLLVRPGLQ CGCCCGGGCCTGCAG SS- BacillusATGAAACAGCAGAAACGCCT 52 MKQQKRLY 114 014 GTATGCGCGCCTGCTGACCCT ARLLTLLFAGCTGTTTGCGCTGATTTTTCTG LIFLLPHSSA CTGCCGCATAGCAGCGCGAGC SA GCG SS-Secretion ATGGCGACGCCGCTGCCTCCG 53 MATPLPPPS 115 015 signalCCCTCCCCGCGGCACCTGCGG PRHLRLLRL CTGCTGCGGCTGCTGCTCTCC LLSGGCCCTCGTCCTCGGC SS- Secretion ATGAAGGCTCCGGGTCGGCTC 54 MKAPGRLV 116 016signal GTGCTCATCATCCTGTGCTCC LIILCSVVFS GTGGTCTTCTCT SS- SecretionATGCTTCAGCTTTGGAAACTT 55 MLQLWKLL 117 017 signal GTTCTCCTGTGCGGCGTGCTCCGVLT ACT SS- Secretion ATGCTTTATCTCCAGGGTTGG 56 MLYLQGWS 118 018 signalAGCATGCCTGCTGTGGCA MPAVA SS- Secretion ATGGATAACGTGCAGCCGAA 57 MDNVQPKI119 019 signal AATAAAACATCGCCCCTTCTG KHRPFCFSV CTTCAGTGTGAAAGGCCACGTKGHVKMLR GAAGATGCTGCGGCTGGATAT LDIINSLVTT TATCAACTCACTGGTAACAACVFMLIVSVL AGTATTCATGCTCATCGTATC ALIP TGTGTTGGCACTGATACCA SS- SecretionATGCCCTGCCTAGACCAACAG 58 MPCLDQQL 120 020 signal CTCACTGTTCATGCCCTACCCTTVHALPCPA GCCCTGCCCAGCCCTCCTCTC QPSSLAFCQ TGGCCTTCTGCCAAGTGGGGT VGFLTATCTTAACAGCA SS- Secretion ATGAAAACCTTGTTCAATCCA 59 MKTLFNPAP 121 021signal GCCCCTGCCATTGCTGACCTG AIADLDPQF GATCCCCAGTTCTACACCCTC YTLSDVFCCTCAGATGTGTTCTGCTGCAAT NESEAEILT GAAAGTGAGGCTGAGATTTTA GLTVGSAAACTGGCCTCACGGTGGGCAGC DA GCTGCAGATGCT SS- SecretionATGAAGCCTCTCCTTGTTGTG 60 MKPLLVVF 122 022 signal TTTGTCTTTCTTTTCCTTTGGGVFLFLWDPV ATCCAGTGCTGGCA LA SS- Secretion ATGTCCTGTTCCCTAAAGTTT 61MSCSLKFTL 123 023 signal ACTTTGATTGTAATTTTTTTTT IVIFFTCTLSACTGTTGGCTTTCATCCAGC SS SS- Secretion ATGGTTCTTACTAAACCTCTTC 62 MVLTKPLQ124 024 signal AAAGAAATGGCAGCATGATG RNGSMMSF AGCTTTGAAAATGTGAAAGAAENVKEKSRE AAGAGCAGAGAAGGAGGGCC GGPHAHTPE CCATGCACACACACCCGAAGA EELCFVVTHAGAATTGTGTTTCGTGGTAAC TPQVQTTLN ACACTACCCTCAGGTTCAGAC LFFHIFKVLTCACACTCAACCTGTTTTTCCAT QPLSLLWG ATATTCAAGGTTCTTACTCAACCACTTTCCCTTCTGTGGGGT SS- Secretion ATGGCCACCCCGCCATTCCGG 63 MATPPFRLI125 025 signal CTGATAAGGAAGATGTTTTCC RKMFSFKVS TTCAAGGTGAGCAGATGGATGRWMGLACF GGGCTTGCCTGCTTCCGGTCC RSLAAS CTGGCGGCATCC SS- SecretionATGAGCTTTTTCCAACTCCTG 64 MSFFQLLM 126 026 signal ATGAAAAGGAAGGAACTCATKRKELIPLV TCCCTTGGTGGTGTTCATGAC VFMTVAAG TGTGGCGGCGGGTGGAGCCTC GASS ATCTSS- Secretion ATGGTCTCAGCTCTGCGGGGA 65 MVSALRGA 127 027 signalGCACCCCTGATCAGGGTGCAC PLIRVHSSPV TCAAGCCCTGTTTCTTCTCCTT SSPSVSGPACTGTGAGTGGACCACGGAGGC ALVSCLSSQ TGGTGAGCTGCCTGTCATCCC SSALSAAAGCTCAGCTCTGAGC SS- Secretion ATGATGGGGTCCCCAGTGAGT 66 MMGSPVSH 128028 signal CATCTGCTGGCCGGCTTCTGT LLAGFCVW GTGTGGGTCGTCTTGGGC VVLG SS-Secretion ATGGCAAGCATGGCTGCCGTG 67 MASMAAVL 129 029 signalCTCACCTGGGCTCTGGCTCTT TWALALLS CTTTCAGCGTTTTCGGCCACC AFSATQA CAGGCA SS-Secretion ATGGTGCTCATGTGGACCAGT 68 MVLMWTSG 130 030 signalGGTGACGCCTTCAAGACGGCC DAFKTAYFL TACTTCCTGCTGAAGGGTGCC LKGAPLQFSCCTCTGCAGTTCTCCGTGTGC VCGLLQVL GGCCTGCTGCAGGTGCTGGTG VDLAILGQAGACCTGGCCATCCTGGGGCAG TA GCCTACGCC SS- Secretion ATGGATTTTGTCGCTGGAGCC69 MDFVAGAI 131 031 signal ATCGGAGGCGTCTGCGGTGTT GGVCGVAVGCTGTGGGCTACCCCCTGGAC GYPLDTVK ACGGTGAAGGTCAGGATCCA VRIQTEPLYGACGGAGCCAAAGTACACAG TGIWHCVR GCATCTGGCACTGCGTCCGGG DTYHRERVATACGTATCACCGAGAGCGCG WGFYRGLS TGTGGG LPVCTVSLV GCTTCTACCGGGGCCTCTCGC SSTGCCCGTGTGCACGGTGTCCC TGGTATCTTCC SS- Secretion ATGGAGAAGCCCCTCTTCCCA 70MEKPLFPLV 132 032 signal TTAGTGCCTTTGCATTGGTTTG PLHWFGFGGCTTTGGCTACACAGCACTGG YTALVVSG TTGTTTCTGGTGGGATCGTTG GIVGYVKTGGCTATGTAAAAACAGGCAGC SVPSLAAGL GTGCCGTCCCTGGCTGCAGGG LFGSLACTGCTCTTCGGCAGTCTAGCC SS- Secretion ATGGGTCTGCTCCTTCCCCTG 71 MGLLLPLAL133 033 signal GCACTCTGCATCCTAGTCCTG CILVLC TGC SS- SecretionATGGGGATCCAGACGAGCCCC 72 MGIQTSPVL 134 034 signal GTCCTGCTGGCCTCCCTGGGGLASLGVGLV GTGGGGCTGGTCACTCTGCTC TLLGLAVG GGCCTGGCTGTGGGC SS- SecretionATGTCGGACCTGCTACTACTG 73 MSDLLLLGL 135 035 signal GGCCTGATTGGGGGCCTGACTIGGLTLLLL CTCTTACTGCTGCTGACGCTG LTLLAFA CTAGCCTTTGCC SS- SecretionATGGAGACTGTGGTGATTGTT 74 METVVIVAI 136 036 signal GCCATAGGTGTGCTGGCCACCGVLATIFLA ATGTTTCTGGCTTCGTTTGCAG SFAALVLVC CCTTGGTGCTGGTTTGCAGGC RQ AGSS- Secretion ATGCGCGGCTCTGTGGAGTGC 75 MAGSVECT 137 037 signalACCTGGGGTTGGGGGCACTGT WGWGHCAP GCCCCCAGCCCCCTGCTCCTT SPLLLWTLLTGGACTCTACTTCTGTTTGCA LFAAPFGLLG GCCCCATTTGGCCTGCTGGGG SS- SecretionATGATGCCGTCCCGTACCAAC 76 MMPSRTNL 138 038 signal CTGGCTACTGGAATCCCCAGTATGIPSSKV AGTAAAGTGAAATATTCAAGG KYSRLSSTD CTCTCCAGCACAGACGATGGCDGYIDLQFK TACATTGACCTTCAGTTTAAG KTPPKIPYK AAAACCCCTCCTAAGATCCCTAIALATVLF TATAAGGCCATCGCACTTGCC LIGA ACTGTGCTGTTTTTGATTGGC GCC SS-Secretion ATGGCCCTGCCCCAGATGTGT 77 MALPQMCD 139 039 signalGACGGGAGCCACTTGGCCTCC GSHLASTLR ACCCTCCGCTATTGCATGACA YCMTVSGTGTCAGCGGCACAGTGGTTCTG VVLVAGTL GTGGCCGGGACGCTCTGCTTC CFA GCT SS- Vrg-6TGAAAAAGTGGTTCGTTGCTG 78 MKKWFVAA 140 041 CCGGCATCGGCGCTGCCGGACGIGAGLLML TCATGCTCTCCAGCGCCGCCA SSAA SS- PhoA ATGAAACAGAGCACCATTGCG 79MKQSTIALA 141 042 CTGGCGCTGCTGCCGCTGCTG LLPLLFTPV TTTACCCCGGTGACCAAAGCGTKA SS- OmpA ATGAAAAAAACCGCGATTGC 80 MKKTAIAIA 142 043GATTGCGGTGGCGCTGGCGGG VALAGFAT CTTTGCGACCGTGGCGCAGGCG VAQA SS- STIATGAAAAAACTGATGCTGGCG 81 MKKLMLAI 143 044 ATTTTTTTTAGCGTGCTGAGCTFFSVLSFPSF TTCCGAGCTTTAGCCAGAGC SQS SS- STII ATGAAAAAAAACATTGCGTTT 82MKKNIAFLL 144 045 CTGCTGGCGAGCATGTTTGTG ASMFVFSIA TTTAGCATTGCGACCAACGCGTNAYA TATGCG SS- Amylase ATGTTTGCGAAACGCTTTAAA 83 MFAKRFKTS 145 046ACCAGCCTGCTGCCGCTGTTT LLPLFAGFL GCGGGCTTTCTGCTGCTGTTTC LLFHLVLAGATCTGGTGCTGGCGGGCCCGG PAAAS CGGCGGCGAGC SS- Alpha ATGCGCTTTCCGAGCATTTTT84 MRFPSIFTA 146 047 Factor ACCGCGGTGCTGTTTGCGGCG VLFAASSALAAGCAGCGCGCTGGCG SS- Alpha ATGCGCTTTCCGAGCATTTTT 85 MRFPSIFTT 147 048Factor ACCACCGTGCTGTTTGCGGCG VLFAASSALA AGCAGCGCGCTGGCG SS- AlphaATGCGCTTTCCGAGCATTTTT 86 MRFPSIFTSV 148 049 Factor ACCAGCGTGCTGTTTGCGGCGLFAASSALA AGCAGCGCGCTGGCG SS- Alpha ATGCGCTTTCCGAGCATTTTT 87 MRFPSIFTH149 050 Factor ACCCATGTGCTGTTTGCGGCG VLFAASSALA AGCAGCGCGCTGGCG SS-Alpha ATGCGCTTTCCGAGCATTTTT 88 MRFPSIFTIV 150 051 FactorACCATTGTGCTGTTTGCGGCG LFAASSALA AGCAGCGCGCTGGCG SS- AlphaATGCGCTTTCCGAGCATTTTT 89 MRFPSIFTFV 151 052 Factor ACCTTTGTGCTGTTTGCGGCGLFAASSALA AGCAGCGCGCTGGCG SS- Alpha ATGCGCTTTCCGAGCATTTTT 90 MRFPSIFTE152 053 Factor ACCGAAGTGCTGTTTGCGGCG VLFAASSALA AGCAGCGCGCTGGCG SS-Alpha ATGCGCTTTCCGAGCATTTTT 91 MRFPSIFTG 153 054 FactorACCGGCGTGCTGTTTGCGGCG VLFAASSALA AGCAGCGCGCTGGCG SS- Endoglucanase VATGCGTTCCTCCCCCCTCCTCC 92 MRSSPLLRS 154 055 GCTCCGCCGTTGTGGCCGCCCAVVAALPV TGCCGGTGTTGGCCCTTGCC LALA SS- Secretion ATGGGCGCGGCGGCCGTGCGC93 MGAAAVR 155 056 signal TGGCACTTGTGCGTGCTGCTG WHLCVLLAGCCCTGGGCACACGCGGGCG LGTRGRL GCTG SS- Fungal ATGAGGAGCTCCCTTGTGCTG 94MRSSLVLFF 156 057 TTCTTTGTCTCTGCGTGGACG VSAWTALA GCCTTGGCCAG SS-Fibronectin ATGCTCAGGGGTCCGGGACCC 95 MLRGPGPG 157 058GGGCGGCTGCTGCTGCTAGCA RLLLLAVLC GTCCTGTGCCTGGGGACATCG LGTSVRCTEGTGCGCTGCACCGAAACCGGG TGKSKR AAGAGCAAGAGG SS- FibronectinATGCTTAGGGGTCCGGGGCCC 96 MLRGPGPG 158 059 GGGCTGCTGCTGCTGGCCGTCLLLLAVQCL CAGCTGGGGACAGCGGTGCCC GTAVPSTGA TCCACG SS- FibronectinATGCGCCGGGGGGCCCTGACC 97 MRRGALTG 159 060 GGGCTGCTCCTGGTCCTGTGCLLLVLCLSV CTGAGTGTTGTGCTACGTGCA VLRAAPSAT GCCCCCTCTGCAACAAGCAAG SKKRRAAGCGCAGG

In table 9, SS is secretion signal and MLS is mitochondrial leadersignal. The primary constructs or mmRNA of the present invention may bedesigned to encode any of the signal peptide sequences of SEQ ID NOs98-159, or fragments or variants thereof. These sequences may beincluded at the beginning of the polypeptide coding region, in themiddle or at the terminus or alternatively into a flanking region.Further, any of the polynucleotide primary constructs of the presentinvention may also comprise one or more of the sequences defined by SEQID NOs 36-97. These may be in the first region or either flankingregion.

Additional signal peptide sequences which may be utilized in the presentinvention include those taught in, for example, databases such as thosefound at http://www.signalpeptide.de/ orhttp://proline.bic.nus.edu.sg/spdb/. Those described in U.S. Pat. Nos.8,124,379; 7,413,875 and 7,385,034 are also within the scope of theinvention and the contents of each are incorporated herein by referencein their entirety.

In one embodiment, the modified nucleic acid molecules may include anucleic acid sequence encoding a nuclear localization signal (NLS)and/or a nuclear export signal (NES). In one aspect, a modified nucleicacid molecules may include a nucleic acid sequence encoding a nuclearlocalization signal (NLS). The modified nucleic acid molecules encodinga NLS would be able to traffic a polypeptide into the nucleus anddeliver a survival or death signal to the nuclear microenvironment. Inanother aspect, the modified nucleic acid molecules may include anucleic acid sequence encoding a nuclear export signal such as NES1and/or NES2. As a nonlimiting example, the modified nucleic acidmolecules may encode a NES1, NES2 and a NLS signal and an oncologyrelated polypeptide or a scrambled sequence which is not translatable inorder to interact with HIF1-alpha to alter the transcritome of thecancer cells.

Target Selection

According to the present invention, the primary constructs comprise atleast a first region of linked nucleosides encoding at least onepolypeptide of interest. The polypeptides of interest or “targets” orproteins and peptides of the present invention are listed in U.S.Provisional Patent Application No. 61/618,862, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Biologics; U.S.Provisional Patent Application No. 61/681,645, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Biologics; U.S.Provisional Patent Application No. 61/737,130, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Biologics; U.S.Provisional Patent Application No. 61/618,866, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Antibodies; U.S.Provisional Patent Application No. 61/681,647, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Antibodies; U.S.Provisional Patent Application No. 61/737,134, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Antibodies; U.S.Provisional Patent Application No. 61/618,868, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Vaccines; U.S.Provisional Patent Application No. 61/681,648, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Vaccines; U.S.Provisional Patent Application No. 61/737,135, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Vaccines; U.S.Provisional Patent Application No. 61/618,870, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of TherapeuticProteins and Peptides; U.S. Provisional Patent Application No.61/681,649, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Therapeutic Proteins and Peptides; U.S. ProvisionalPatent Application No. 61/737,139, filed Dec. 14, 2012, ModifiedPolynucleotides for the Production of Therapeutic Proteins and Peptides;U.S. Provisional Patent Application No. 61/618,873, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of SecretedProteins; U.S. Provisional Patent Application No. 61/681,650, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins; U.S. Provisional Patent Application No. 61/737,147,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Secreted Proteins; U.S. Provisional Patent Application No.61/618,878, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Plasma Membrane Proteins; U.S. Provisional PatentApplication No. 61/681,654, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Plasma Membrane Proteins; U.S.Provisional Patent Application No. 61/737,152, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins; U.S. Provisional Patent Application No. 61/618,885, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins; U.S. Provisional PatentApplication No. 61/681,658, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins; U.S. Provisional Patent Application No. 61/737,155, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins; U.S. Provisional PatentApplication No. 61/618,896, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins; U.S. Provisional Patent Application No. 61/668,157, filed Jul.5, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins; U.S. Provisional PatentApplication No. 61/681,661, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins; U.S. Provisional Patent Application No. 61/737,160, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins; U.S. Provisional PatentApplication No. 61/618,911, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Nuclear Proteins; U.S. ProvisionalPatent Application No. 61/681,667, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Nuclear Proteins; U.S.Provisional Patent Application No. 61/737,168, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins; U.S. Provisional Patent Application No. 61/618,922, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins; U.S. Provisional Patent Application No. 61/681,675, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins; U.S. Provisional Patent Application No. 61/737,174, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins; U.S. Provisional Patent Application No. 61/618,935, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/681,687, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/737,184, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/618,945, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/681,696, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/737,191, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/618,953, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease; U.S. Provisional PatentApplication No. 61/681,704, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; U.S. Provisional Patent Application No. 61/737,203, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/753,661, entitled Polynucleotides For The AlterationOf Cellular Phenotypes And Microenvironments; International ApplicationNo PCT/US2013/030062, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Biologics and Proteins Associatedwith Human Disease; International Application No PCT/US2013/030063,filed Mar. 9, 2013, entitled Modified Polynucleotides; InternationalApplication No. PCT/US2013/030064, entitled Modified Polynucleotides forthe Production of Secreted Proteins; International Application NoPCT/US2013/030059, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Membrane Proteins; International Application No.PCT/US2013/030066, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Cytoplasmic and Cytoskeletal Proteins;International Application No. PCT/US2013/030067, filed Mar. 9, 2013,entitled Modified Polynucleotides for the Production of NuclearProteins; International Application No. PCT/US2013/030060, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of Proteins;International Application No. PCT/US2013/030061, filed Mar. 9, 2013,entitled Modified Polynucleotides for the Production of ProteinsAssociated with Human Disease; International Application No.PCT/US2013/030068, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Cosmetic Proteins and Peptides; InternationalApplication No. PCT/US2013/030070, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Oncology-Related Proteins andPeptides; and International Application No. PCT/US2013/031821, filedMar. 15, 2013, entitled In Vivo Production of Proteins, the contents ofeach of which are herein incorporated by reference in their entireties.

Protein Cleavage Signals and Sites

In one embodiment, the polypeptides of the present invention may includeat least one protein cleavage signal containing at least one proteincleavage site. The protein cleavage site may be located at theN-terminus, the C-terminus, at any space between the N- and theC-termini such as, but not limited to, half-way between the N- andC-termini, between the N-terminus and the half way point, between thehalf way point and the C-terminus, and combinations thereof.

The polypeptides of the present invention may include, but is notlimited to, a proprotein convertase (or prohormone convertase), thrombinor Factor Xa protein cleavage signal. Proprotein convertases are afamily of nine proteinases, comprising seven basic amino acid-specificsubtilisin-like serine proteinases related to yeast kexin, known asprohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basicamino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilasesthat cleave at non-basic residues, called subtilisin kexin isozyme 1(SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9).Non-limiting examples of protein cleavage signal amino acid sequencesare listing in Table 10. In Table 10, “X” refers to any amino acid, “n”may be 0, 2, 4 or 6 amino acids and “*” refers to the protein cleavagesite. In Table 10, SEQ ID NO: 162 refers to when n=4 and SEQ ID NO:163refers to when n=6.

TABLE 10 Protein Cleavage Site Sequences Protein Cleavage Amino AcidSignal Cleavage Sequence SEQ ID NO Proprotein R-X-X-R* 160 convertaseR-X-K/R-R* 161 K/R-Xn-K/R* 162 or 163 Thrombin L-V-P-R*-G-S 164 L-V-P-R*165 A/F/G/I/L/T/V/M-A/F/G/ 166 I/L/T/V/W/A-P-R* Factor Xa I-E-G-R* 167I-D-G-R* 168 A-E-G-R* 169 A/F/G/I/L/T/V/M-D/E- 170 G-R*

In one embodiment, the primary constructs, modified nucleic acids andthe mmRNA of the present invention may be engineered such that theprimary construct, modified nucleic acid or mmRNA contains at least oneencoded protein cleavage signal. The encoded protein cleavage signal maybe located before the start codon, after the start codon, before thecoding region, within the coding region such as, but not limited to,half way in the coding region, between the start codon and the half waypoint, between the half way point and the stop codon, after the codingregion, before the stop codon, between two stop codons, after the stopcodon and combinations thereof.

In one embodiment, the primary constructs, modified nucleic acids ormmRNA of the present invention may include at least one encoded proteincleavage signal containing at least one protein cleavage site. Theencoded protein cleavage signal may include, but is not limited to, aproprotein convertase (or prohormone convertase), thrombin and/or FactorXa protein cleavage signal. One of skill in the art may use Table 5above or other known methods to determine the appropriate encodedprotein cleavage signal to include in the primary constructs, modifiednucleic acids or mmRNA of the present invention. For example, startingwith the signal of Table 10 and considering the codons of Table 5 onecan design a signal for the primary construct which can produce aprotein signal in the resulting polypeptide.

In one embodiment, the polypeptides of the present invention include atleast one protein cleavage signal and/or site.

As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Pub. No.20090227660, herein incorporated by reference in their entireties, use afurin cleavage site to cleave the N-terminal methionine of GLP-1 in theexpression product from the Golgi apparatus of the cells. In oneembodiment, the polypeptides of the present invention include at leastone protein cleavage signal and/or site with the proviso that thepolypeptide is not GLP-1.

In one embodiment, the primary constructs, modified nucleic acids ormmRNA of the present invention includes at least one encoded proteincleavage signal and/or site.

In one embodiment, the primary constructs, modified nucleic acid ormmRNA of the present invention includes at least one encoded proteincleavage signal and/or site with the proviso that the primary construct,modified nucleic acid or mmRNA does not encode GLP-1.

In one embodiment, the primary constructs, modified nucleic acid ormmRNA of the present invention may include more than one coding region.Where multiple coding regions are present in the primary construct,modified nucleic acid or mmRNA of the present invention, the multiplecoding regions may be separated by encoded protein cleavage sites. As anon-limiting example, the primary construct, modified nucleic acid ormmRNA may be signed in an ordered pattern. On such pattern follows AXBYform where A and B are coding regions which may be the same or differentcoding regions and/or may encode the same or different polypeptides, andX and Y are encoded protein cleavage signals which may encode the sameor different protein cleavage signals. A second such pattern follows theform AXYBZ where A and B are coding regions which may be the same ordifferent coding regions and/or may encode the same or differentpolypeptides, and X, Y and Z are encoded protein cleavage signals whichmay encode the same or different protein cleavage signals. A thirdpattern follows the form ABXCY where A, B and C are coding regions whichmay be the same or different coding regions and/or may encode the sameor different polypeptides, and X and Y are encoded protein cleavagesignals which may encode the same or different protein cleavage signals.

In on embodiment, the polypeptides, primary constructs, modified nucleicacids and mmRNA can also contain sequences that encode protein cleavagesites so that the polypeptides, primary constructs, modified nucleicacids and mmRNA can be released from a carrier region or a fusionpartner by treatment with a specific protease for said protein cleavagesite.

Table 11 is a non-exhaustive listing of miR5 and miR binding sites (miRBS) and their sequences which may be used with the present invention.

TABLE 11 Mirs and mir binding sites mir SEQ MIR BS microRNA ID SEQ IDhsa-let-7a-2-3p 171 1192 hsa-let-7a-3p 172 1193 hsa-let-7a-5p 173 1194hsa-let-7b-3p 174 1195 hsa-let-7b-5p 175 1196 hsa-let-7c 176 1197hsa-let-7d-3p 177 1198 hsa-let-7d-5p 178 1199 hsa-let-7e-3p 179 1200hsa-let-7e-5p 180 1201 hsa-let-7f-1-3p 181 1202 hsa-let-7f-2-3p 182 1203hsa-let-7f-5p 183 1204 hsa-let-7g-3p 184 1205 hsa-let-7g-5p 185 1206hsa-let-7i-3p 186 1207 hsa-let-7i-5p 187 1208 hsa-miR-1 188 1209hsa-miR-100-3p 189 1210 hsa-miR-100-5p 190 1211 hsa-miR-101-3p 191 1212hsa-miR-101-5p 192 1213 hsa-miR-103a-2-5p 193 1214 hsa-miR-103a-3p 1941215 hsa-miR-103b 195 1216 hsa-miR-105-3p 196 1217 hsa-miR-105-5p 1971218 hsa-miR-106a-3p 198 1219 hsa-miR-106a-5p 199 1220 hsa-miR-106b-3p200 1221 hsa-miR-106b-5p 201 1222 hsa-miR-107 202 1223 hsa-miR-10a-3p203 1224 hsa-miR-10a-5p 204 1225 hsa-miR-10b-3p 205 1226 hsa-miR-10b-5p206 1227 hsa-miR-1178-3p 207 1228 hsa-miR-1178-5p 208 1229 hsa-miR-1179209 1230 hsa-miR-1180 210 1231 hsa-miR-1181 211 1232 hsa-miR-1182 2121233 hsa-miR-1183 213 1234 hsa-miR-1184 214 1235 hsa-miR-1185-1-3p 2151236 hsa-miR-1185-2-3p 216 1237 hsa-miR-1185-5p 217 1238 hsa-miR-1193218 1239 hsa-miR-1197 219 1240 hsa-miR-1200 220 1241 hsa-miR-1202 2211242 hsa-miR-1203 222 1243 hsa-miR-1204 223 1244 hsa-miR-1205 224 1245hsa-miR-1206 225 1246 hsa-miR-1207-3p 226 1247 hsa-miR-1207-5p 227 1248hsa-miR-1208 228 1249 hsa-miR-122-3p 229 1250 hsa-miR-1224-3p 230 1251hsa-miR-1224-5p 231 1252 hsa-miR-1225-3p 232 1253 hsa-miR-1225-5p 2331254 hsa-miR-122-5p 234 1255 hsa-miR-1226-3p 235 1256 hsa-miR-1226-5p236 1257 hsa-miR-1227-3p 237 1258 hsa-miR-1227-5p 238 1259hsa-miR-1228-3p 239 1260 hsa-miR-1228-5p 240 1261 hsa-miR-1229-3p 2411262 hsa-miR-1229-5p 242 1263 hsa-miR-1231 243 1264 hsa-miR-1233-1-5p244 1265 hsa-miR-1233-3p 245 1266 hsa-miR-1234-3p 246 1267hsa-miR-1234-5p 247 1268 hsa-miR-1236-3p 248 1269 hsa-miR-1236-5p 2491270 hsa-miR-1237-3p 250 1271 hsa-miR-1237-5p 251 1272 hsa-miR-1238-3p252 1273 hsa-miR-1238-5p 253 1274 hsa-miR-1243 254 1275 hsa-miR-124-3p255 1276 hsa-miR-1244 256 1277 hsa-miR-1245a 257 1278 hsa-miR-1245b-3p258 1279 hsa-miR-1245b-5p 259 1280 hsa-miR-124-5p 260 1281 hsa-miR-1246261 1282 hsa-miR-1247-3p 262 1283 hsa-miR-1247-5p 263 1284 hsa-miR-1248264 1285 hsa-miR-1249 265 1286 hsa-miR-1250 266 1287 hsa-miR-1251 2671288 hsa-miR-1252 268 1289 hsa-miR-1253 269 1290 hsa-miR-1254 270 1291hsa-miR-1255a 271 1292 hsa-miR-1255b-2-3p 272 1293 hsa-miR-1255b-5p 2731294 hsa-miR-1256 274 1295 hsa-miR-1257 275 1296 hsa-miR-1258 276 1297hsa-miR-125a-3p 277 1298 hsa-miR-125a-5p 278 1299 hsa-miR-125b-1-3p 2791300 hsa-miR-125b-2-3p 280 1301 hsa-miR-125b-5p 281 1302 hsa-miR-1260a282 1303 hsa-miR-1260b 283 1304 hsa-miR-1261 284 1305 hsa-miR-1262 2851306 hsa-miR-1263 286 1307 hsa-miR-126-3p 287 1308 hsa-miR-1264 288 1309hsa-miR-1265 289 1310 hsa-miR-126-5p 290 1311 hsa-miR-1266 291 1312hsa-miR-1267 292 1313 hsa-miR-1268a 293 1314 hsa-miR-1268b 294 1315hsa-miR-1269a 295 1316 hsa-miR-1269b 296 1317 hsa-miR-1270 297 1318hsa-miR-1271-3p 298 1319 hsa-miR-1271-5p 299 1320 hsa-miR-1272 300 1321hsa-miR-1273a 301 1322 hsa-miR-1273c 302 1323 hsa-miR-1273d 303 1324hsa-miR-1273e 304 1325 hsa-miR-1273f 305 1326 hsa-miR-1273g-3p 306 1327hsa-miR-1273g-5p 307 1328 hsa-miR-127-3p 308 1329 hsa-miR-1275 309 1330hsa-miR-127-5p 310 1331 hsa-miR-1276 311 1332 hsa-miR-1277-3p 312 1333hsa-miR-1277-5p 313 1334 hsa-miR-1278 314 1335 hsa-miR-1279 315 1336hsa-miR-128 316 1337 hsa-miR-1281 317 1338 hsa-miR-1282 318 1339hsa-miR-1283 319 1340 hsa-miR-1284 320 1341 hsa-miR-1285-3p 321 1342hsa-miR-1285-5p 322 1343 hsa-miR-1286 323 1344 hsa-miR-1287 324 1345hsa-miR-1288 325 1346 hsa-miR-1289 326 1347 hsa-miR-1290 327 1348hsa-miR-1291 328 1349 hsa-miR-129-1-3p 329 1350 hsa-miR-1292-3p 330 1351hsa-miR-129-2-3p 331 1352 hsa-miR-1292-5p 332 1353 hsa-miR-1293 333 1354hsa-miR-1294 334 1355 hsa-miR-1295a 335 1356 hsa-miR-1295b-3p 336 1357hsa-miR-1295b-5p 337 1358 hsa-miR-129-5p 338 1359 hsa-miR-1296 339 1360hsa-miR-1297 340 1361 hsa-miR-1298 341 1362 hsa-miR-1299 342 1363hsa-miR-1301 343 1364 hsa-miR-1302 344 1365 hsa-miR-1303 345 1366hsa-miR-1304-3p 346 1367 hsa-miR-1304-5p 347 1368 hsa-miR-1305 348 1369hsa-miR-1306-3p 349 1370 hsa-miR-1306-5p 350 1371 hsa-miR-1307-3p 3511372 hsa-miR-1307-5p 352 1373 hsa-miR-130a-3p 353 1374 hsa-miR-130a-5p354 1375 hsa-miR-130b-3p 355 1376 hsa-miR-130b-5p 356 1377 hsa-miR-1321357 1378 hsa-miR-1322 358 1379 hsa-miR-1323 359 1380 hsa-miR-132-3p 3601381 hsa-miR-1324 361 1382 hsa-miR-132-5p 362 1383 hsa-miR-133a 363 1384hsa-miR-133b 364 1385 hsa-miR-134 365 1386 hsa-miR-1343 366 1387hsa-miR-135a-3p 367 1388 hsa-miR-135a-5p 368 1389 hsa-miR-135b-3p 3691390 hsa-miR-135b-5p 370 1391 hsa-miR-136-3p 371 1392 hsa-miR-136-5p 3721393 hsa-miR-137 373 1394 hsa-miR-138-1-3p 374 1395 hsa-miR-138-2-3p 3751396 hsa-miR-138-5p 376 1397 hsa-miR-139-3p 377 1398 hsa-miR-139-5p 3781399 hsa-miR-140-3p 379 1400 hsa-miR-140-5p 380 1401 hsa-miR-141-3p 3811402 hsa-miR-141-5p 382 1403 hsa-miR-142-3p 383 1404 hsa-miR-142-5p 3841405 hsa-miR-143-3p 385 1406 hsa-miR-143-5p 386 1407 hsa-miR-144-3p 3871408 hsa-miR-144-5p 388 1409 hsa-miR-145-3p 389 1410 hsa-miR-145-5p 3901411 hsa-miR-1468 391 1412 hsa-miR-1469 392 1413 hsa-miR-146a-3p 3931414 hsa-miR-146a-5p 394 1415 hsa-miR-146b-3p 395 1416 hsa-miR-146b-5p396 1417 hsa-miR-1470 397 1418 hsa-miR-1471 398 1419 hsa-miR-147a 3991420 hsa-miR-147b 400 1421 hsa-miR-148a-3p 401 1422 hsa-miR-148a-5p 4021423 hsa-miR-148b-3p 403 1424 hsa-miR-148b-5p 404 1425 hsa-miR-149-3p405 1426 hsa-miR-149-5p 406 1427 hsa-miR-150-3p 407 1428 hsa-miR-150-5p408 1429 hsa-miR-151a-3p 409 1430 hsa-miR-151a-5p 410 1431 hsa-miR-151b411 1432 hsa-miR-152 412 1433 hsa-miR-153 413 1434 hsa-miR-1537 414 1435hsa-miR-1538 415 1436 hsa-miR-1539 416 1437 hsa-miR-154-3p 417 1438hsa-miR-154-5p 418 1439 hsa-miR-155-3p 419 1440 hsa-miR-155-5p 420 1441hsa-miR-1587 421 1442 hsa-miR-15a-3p 422 1443 hsa-miR-15a-5p 423 1444hsa-miR-15b-3p 424 1445 hsa-miR-15b-5p 425 1446 hsa-miR-16-1-3p 426 1447hsa-miR-16-2-3p 427 1448 hsa-miR-16-5p 428 1449 hsa-miR-17-3p 429 1450hsa-miR-17-5p 430 1451 hsa-miR-181a-2-3p 431 1452 hsa-miR-181a-3p 4321453 hsa-miR-181a-5p 433 1454 hsa-miR-181b-3p 434 1455 hsa-miR-181b-5p435 1456 hsa-miR-181c-3p 436 1457 hsa-miR-181c-5p 437 1458 hsa-miR-181d438 1459 hsa-miR-182-3p 439 1460 hsa-miR-1825 440 1461 hsa-miR-182-5p441 1462 hsa-miR-1827 442 1463 hsa-miR-183-3p 443 1464 hsa-miR-183-5p444 1465 hsa-miR-184 445 1466 hsa-miR-185-3p 446 1467 hsa-miR-185-5p 4471468 hsa-miR-186-3p 448 1469 hsa-miR-186-5p 449 1470 hsa-miR-187-3p 4501471 hsa-miR-187-5p 451 1472 hsa-miR-188-3p 452 1473 hsa-miR-188-5p 4531474 hsa-miR-18a-3p 454 1475 hsa-miR-18a-5p 455 1476 hsa-miR-18b-3p 4561477 hsa-miR-18b-5p 457 1478 hsa-miR-1908 458 1479 hsa-miR-1909-3p 4591480 hsa-miR-1909-5p 460 1481 hsa-miR-190a 461 1482 hsa-miR-190b 4621483 hsa-miR-1910 463 1484 hsa-miR-1911-3p 464 1485 hsa-miR-1911-5p 4651486 hsa-miR-1912 466 1487 hsa-miR-1913 467 1488 hsa-miR-191-3p 468 1489hsa-miR-1914-3p 469 1490 hsa-miR-1914-5p 470 1491 hsa-miR-1915-3p 4711492 hsa-miR-1915-5p 472 1493 hsa-miR-191-5p 473 1494 hsa-miR-192-3p 4741495 hsa-miR-192-5p 475 1496 hsa-miR-193a-3p 476 1497 hsa-miR-193a-5p477 1498 hsa-miR-193b-3p 478 1499 hsa-miR-193b-5p 479 1500hsa-miR-194-3p 480 1501 hsa-miR-194-5p 481 1502 hsa-miR-195-3p 482 1503hsa-miR-195-5p 483 1504 hsa-miR-196a-3p 484 1505 hsa-miR-196a-5p 4851506 hsa-miR-196b-3p 486 1507 hsa-miR-196b-5p 487 1508 hsa-miR-1972 4881509 hsa-miR-1973 489 1510 hsa-miR-197-3p 490 1511 hsa-miR-197-5p 4911512 hsa-miR-1976 492 1513 hsa-miR-198 493 1514 hsa-miR-199a-3p 494 1515hsa-miR-199a-5p 495 1516 hsa-miR-199b-3p 496 1517 hsa-miR-199b-5p 4971518 hsa-miR-19a-3p 498 1519 hsa-miR-19a-5p 499 1520 hsa-miR-19b-1-5p500 1521 hsa-miR-19b-2-5p 501 1522 hsa-miR-19b-3p 502 1523hsa-miR-200a-3p 503 1524 hsa-miR-200a-5p 504 1525 hsa-miR-200b-3p 5051526 hsa-miR-200b-5p 506 1527 hsa-miR-200c-3p 507 1528 hsa-miR-200c-5p508 1529 hsa-miR-202-3p 509 1530 hsa-miR-202-5p 510 1531 hsa-miR-203a511 1532 hsa-miR-203b-3p 512 1533 hsa-miR-203b-5p 513 1534hsa-miR-204-3p 514 1535 hsa-miR-204-5p 515 1536 hsa-miR-2052 516 1537hsa-miR-2053 517 1538 hsa-miR-205-3p 518 1539 hsa-miR-2054 519 1540hsa-miR-205-5p 520 1541 hsa-miR-206 521 1542 hsa-miR-208a 522 1543hsa-miR-208b 523 1544 hsa-miR-20a-3p 524 1545 hsa-miR-20a-5p 525 1546hsa-miR-20b-3p 526 1547 hsa-miR-20b-5p 527 1548 hsa-miR-210 528 1549hsa-miR-2110 529 1550 hsa-miR-2113 530 1551 hsa-miR-211-3p 531 1552hsa-miR-2114-3p 532 1553 hsa-miR-2114-5p 533 1554 hsa-miR-2115-3p 5341555 hsa-miR-2115-5p 535 1556 hsa-miR-211-5p 536 1557 hsa-miR-2116-3p537 1558 hsa-miR-2116-5p 538 1559 hsa-miR-2117 539 1560 hsa-miR-212-3p540 1561 hsa-miR-212-5p 541 1562 hsa-miR-21-3p 542 1563 hsa-miR-214-3p543 1564 hsa-miR-214-5p 544 1565 hsa-miR-215 545 1566 hsa-miR-21-5p 5461567 hsa-miR-216a-3p 547 1568 hsa-miR-216a-5p 548 1569 hsa-miR-216b 5491570 hsa-miR-217 550 1571 hsa-miR-218-1-3p 551 1572 hsa-miR-218-2-3p 5521573 hsa-miR-218-5p 553 1574 hsa-miR-219-1-3p 554 1575 hsa-miR-219-2-3p555 1576 hsa-miR-219-5p 556 1577 hsa-miR-221-3p 557 1578 hsa-miR-221-5p558 1579 hsa-miR-222-3p 559 1580 hsa-miR-222-5p 560 1581 hsa-miR-223-3p561 1582 hsa-miR-223-5p 562 1583 hsa-miR-22-3p 563 1584 hsa-miR-224-3p564 1585 hsa-miR-224-5p 565 1586 hsa-miR-22-5p 566 1587 hsa-miR-2276 5671588 hsa-miR-2277-3p 568 1589 hsa-miR-2277-5p 569 1590 hsa-miR-2278 5701591 hsa-miR-2355-3p 571 1592 hsa-miR-2355-5p 572 1593 hsa-miR-2392 5731594 hsa-miR-23a-3p 574 1595 hsa-miR-23a-5p 575 1596 hsa-miR-23b-3p 5761597 hsa-miR-23b-5p 577 1598 hsa-miR-23c 578 1599 hsa-miR-24-1-5p 5791600 hsa-miR-24-2-5p 580 1601 hsa-miR-24-3p 581 1602 hsa-miR-2467-3p 5821603 hsa-miR-2467-5p 583 1604 hsa-miR-25-3p 584 1605 hsa-miR-25-5p 5851606 hsa-miR-2681-3p 586 1607 hsa-miR-2681-5p 587 1608 hsa-miR-2682-3p588 1609 hsa-miR-2682-5p 589 1610 hsa-miR-26a-1-3p 590 1611hsa-miR-26a-2-3p 591 1612 hsa-miR-26a-5p 592 1613 hsa-miR-26b-3p 5931614 hsa-miR-26b-5p 594 1615 hsa-miR-27a-3p 595 1616 hsa-miR-27a-5p 5961617 hsa-miR-27b-3p 597 1618 hsa-miR-27b-5p 598 1619 hsa-miR-28-3p 5991620 hsa-miR-28-5p 600 1621 hsa-miR-2861 601 1622 hsa-miR-2909 602 1623hsa-miR-296-3p 603 1624 hsa-miR-2964a-3p 604 1625 hsa-miR-2964a-5p 6051626 hsa-miR-296-5p 606 1627 hsa-miR-297 607 1628 hsa-miR-298 608 1629hsa-miR-299-3p 609 1630 hsa-miR-299-5p 610 1631 hsa-miR-29a-3p 611 1632hsa-miR-29a-5p 612 1633 hsa-miR-29b-1-5p 613 1634 hsa-miR-29b-2-5p 6141635 hsa-miR-29b-3p 615 1636 hsa-miR-29c-3p 616 1637 hsa-miR-29c-5p 6171638 hsa-miR-300 618 1639 hsa-miR-301a-3p 619 1640 hsa-miR-301a-5p 6201641 hsa-miR-301b 621 1642 hsa-miR-302a-3p 622 1643 hsa-miR-302a-5p 6231644 hsa-miR-302b-3p 624 1645 hsa-miR-302b-5p 625 1646 hsa-miR-302c-3p626 1647 hsa-miR-302c-5p 627 1648 hsa-miR-302d-3p 628 1649hsa-miR-302d-5p 629 1650 hsa-miR-302e 630 1651 hsa-miR-302f 631 1652hsa-miR-3064-3p 632 1653 hsa-miR-3064-5p 633 1654 hsa-miR-3065-3p 6341655 hsa-miR-3065-5p 635 1656 hsa-miR-3074-3p 636 1657 hsa-miR-3074-5p637 1658 hsa-miR-30a-3p 638 1659 hsa-miR-30a-5p 639 1660 hsa-miR-30b-3p640 1661 hsa-miR-30b-5p 641 1662 hsa-miR-30c-1-3p 642 1663hsa-miR-30c-2-3p 643 1664 hsa-miR-30c-5p 644 1665 hsa-miR-30d-3p 6451666 hsa-miR-30d-5p 646 1667 hsa-miR-30e-3p 647 1668 hsa-miR-30e-5p 6481669 hsa-miR-3115 649 1670 hsa-miR-3116 650 1671 hsa-miR-3117-3p 6511672 hsa-miR-3117-5p 652 1673 hsa-miR-3118 653 1674 hsa-miR-3119 6541675 hsa-miR-3120-3p 655 1676 hsa-miR-3120-5p 656 1677 hsa-miR-3121-3p657 1678 hsa-miR-3121-5p 658 1679 hsa-miR-3122 659 1680 hsa-miR-3123 6601681 hsa-miR-3124-3p 661 1682 hsa-miR-3124-5p 662 1683 hsa-miR-3125 6631684 hsa-miR-3126-3p 664 1685 hsa-miR-3126-5p 665 1686 hsa-miR-3127-3p666 1687 hsa-miR-3127-5p 667 1688 hsa-miR-3128 668 1689 hsa-miR-3129-3p669 1690 hsa-miR-3129-5p 670 1691 hsa-miR-3130-3p 671 1692hsa-miR-3130-5p 672 1693 hsa-miR-3131 673 1694 hsa-miR-3132 674 1695hsa-miR-3133 675 1696 hsa-miR-3134 676 1697 hsa-miR-3135a 677 1698hsa-miR-3135b 678 1699 hsa-miR-3136-3p 679 1700 hsa-miR-3136-5p 680 1701hsa-miR-3137 681 1702 hsa-miR-3138 682 1703 hsa-miR-3139 683 1704hsa-miR-31-3p 684 1705 hsa-miR-3140-3p 685 1706 hsa-miR-3140-5p 686 1707hsa-miR-3141 687 1708 hsa-miR-3142 688 1709 hsa-miR-3143 689 1710hsa-miR-3144-3p 690 1711 hsa-miR-3144-5p 691 1712 hsa-miR-3145-3p 6921713 hsa-miR-3145-5p 693 1714 hsa-miR-3146 694 1715 hsa-miR-3147 6951716 hsa-miR-3148 696 1717 hsa-miR-3149 697 1718 hsa-miR-3150a-3p 6981719 hsa-miR-3150a-5p 699 1720 hsa-miR-3150b-3p 700 1721hsa-miR-3150b-5p 701 1722 hsa-miR-3151 702 1723 hsa-miR-3152-3p 703 1724hsa-miR-3152-5p 704 1725 hsa-miR-3153 705 1726 hsa-miR-3154 706 1727hsa-miR-3155a 707 1728 hsa-miR-3155b 708 1729 hsa-miR-3156-3p 709 1730hsa-miR-3156-5p 710 1731 hsa-miR-3157-3p 711 1732 hsa-miR-3157-5p 7121733 hsa-miR-3158-3p 713 1734 hsa-miR-3158-5p 714 1735 hsa-miR-3159 7151736 hsa-miR-31-5p 716 1737 hsa-miR-3160-3p 717 1738 hsa-miR-3160-5p 7181739 hsa-miR-3161 719 1740 hsa-miR-3162-3p 720 1741 hsa-miR-3162-5p 7211742 hsa-miR-3163 722 1743 hsa-miR-3164 723 1744 hsa-miR-3165 724 1745hsa-miR-3166 725 1746 hsa-miR-3167 726 1747 hsa-miR-3168 727 1748hsa-miR-3169 728 1749 hsa-miR-3170 729 1750 hsa-miR-3171 730 1751hsa-miR-3173-3p 731 1752 hsa-miR-3173-5p 732 1753 hsa-miR-3174 733 1754hsa-miR-3175 734 1755 hsa-miR-3176 735 1756 hsa-miR-3177-3p 736 1757hsa-miR-3177-5p 737 1758 hsa-miR-3178 738 1759 hsa-miR-3179 739 1760hsa-miR-3180 740 1761 hsa-miR-3180-3p 741 1762 hsa-miR-3180-5p 742 1763hsa-miR-3181 743 1764 hsa-miR-3182 744 1765 hsa-miR-3183 745 1766hsa-miR-3184-3p 746 1767 hsa-miR-3184-5p 747 1768 hsa-miR-3185 748 1769hsa-miR-3186-3p 749 1770 hsa-miR-3186-5p 750 1771 hsa-miR-3187-3p 7511772 hsa-miR-3187-5p 752 1773 hsa-miR-3188 753 1774 hsa-miR-3189-3p 7541775 hsa-miR-3189-5p 755 1776 hsa-miR-3190-3p 756 1777 hsa-miR-3190-5p757 1778 hsa-miR-3191-3p 758 1779 hsa-miR-3191-5p 759 1780 hsa-miR-3192760 1781 hsa-miR-3193 761 1782 hsa-miR-3194-3p 762 1783 hsa-miR-3194-5p763 1784 hsa-miR-3195 764 1785 hsa-miR-3196 765 1786 hsa-miR-3197 7661787 hsa-miR-3198 767 1788 hsa-miR-3199 768 1789 hsa-miR-3200-3p 7691790 hsa-miR-3200-5p 770 1791 hsa-miR-3201 771 1792 hsa-miR-3202 7721793 hsa-miR-320a 773 1794 hsa-miR-320b 774 1795 hsa-miR-320c 775 1796hsa-miR-320d 776 1797 hsa-miR-320e 777 1798 hsa-miR-323a-3p 778 1799hsa-miR-323a-5p 779 1800 hsa-miR-323b-3p 780 1801 hsa-miR-323b-5p 7811802 hsa-miR-32-3p 782 1803 hsa-miR-324-3p 783 1804 hsa-miR-324-5p 7841805 hsa-miR-325 785 1806 hsa-miR-32-5p 786 1807 hsa-miR-326 787 1808hsa-miR-328 788 1809 hsa-miR-329 789 1810 hsa-miR-330-3p 790 1811hsa-miR-330-5p 791 1812 hsa-miR-331-3p 792 1813 hsa-miR-331-5p 793 1814hsa-miR-335-3p 794 1815 hsa-miR-335-5p 795 1816 hsa-miR-337-3p 796 1817hsa-miR-337-5p 797 1818 hsa-miR-338-3p 798 1819 hsa-miR-338-5p 799 1820hsa-miR-339-3p 800 1821 hsa-miR-339-5p 801 1822 hsa-miR-33a-3p 802 1823hsa-miR-33a-5p 803 1824 hsa-miR-33b-3p 804 1825 hsa-miR-33b-5p 805 1826hsa-miR-340-3p 806 1827 hsa-miR-340-5p 807 1828 hsa-miR-342-3p 808 1829hsa-miR-342-5p 809 1830 hsa-miR-345-3p 810 1831 hsa-miR-345-5p 811 1832hsa-miR-346 812 1833 hsa-miR-34a-3p 813 1834 hsa-miR-34a-5p 814 1835hsa-miR-34b-3p 815 1836 hsa-miR-34b-5p 816 1837 hsa-miR-34c-3p 817 1838hsa-miR-34c-5p 818 1839 hsa-miR-3529-3p 819 1840 hsa-miR-3529-5p 8201841 hsa-miR-3591-3p 821 1842 hsa-miR-3591-5p 822 1843 hsa-miR-3605-3p823 1844 hsa-miR-3605-5p 824 1845 hsa-miR-3606-3p 825 1846hsa-miR-3606-5p 826 1847 hsa-miR-3607-3p 827 1848 hsa-miR-3607-5p 8281849 hsa-miR-3609 829 1850 hsa-miR-3610 830 1851 hsa-miR-3611 831 1852hsa-miR-3612 832 1853 hsa-miR-3613-3p 833 1854 hsa-miR-3613-5p 834 1855hsa-miR-361-3p 835 1856 hsa-miR-3614-3p 836 1857 hsa-miR-3614-5p 8371858 hsa-miR-3615 838 1859 hsa-miR-361-5p 839 1860 hsa-miR-3616-3p 8401861 hsa-miR-3616-5p 841 1862 hsa-miR-3617-3p 842 1863 hsa-miR-3617-5p843 1864 hsa-miR-3618 844 1865 hsa-miR-3619-3p 845 1866 hsa-miR-3619-5p846 1867 hsa-miR-3620-3p 847 1868 hsa-miR-3620-5p 848 1869 hsa-miR-3621849 1870 hsa-miR-3622a-3p 850 1871 hsa-miR-3622a-5p 851 1872hsa-miR-3622b-3p 852 1873 hsa-miR-3622b-5p 853 1874 hsa-miR-362-3p 8541875 hsa-miR-362-5p 855 1876 hsa-miR-363-3p 856 1877 hsa-miR-363-5p 8571878 hsa-miR-3646 858 1879 hsa-miR-3648 859 1880 hsa-miR-3649 860 1881hsa-miR-3650 861 1882 hsa-miR-3651 862 1883 hsa-miR-3652 863 1884hsa-miR-3653 864 1885 hsa-miR-3654 865 1886 hsa-miR-3655 866 1887hsa-miR-3656 867 1888 hsa-miR-3657 868 1889 hsa-miR-3658 869 1890hsa-miR-3659 870 1891 hsa-miR-365a-3p 871 1892 hsa-miR-365a-5p 872 1893hsa-miR-365b-3p 873 1894 hsa-miR-365b-5p 874 1895 hsa-miR-3660 875 1896hsa-miR-3661 876 1897 hsa-miR-3662 877 1898 hsa-miR-3663-3p 878 1899hsa-miR-3663-5p 879 1900 hsa-miR-3664-3p 880 1901 hsa-miR-3664-5p 8811902 hsa-miR-3665 882 1903 hsa-miR-3666 883 1904 hsa-miR-3667-3p 8841905 hsa-miR-3667-5p 885 1906 hsa-miR-3668 886 1907 hsa-miR-3669 8871908 hsa-miR-3670 888 1909 hsa-miR-3671 889 1910 hsa-miR-3672 890 1911hsa-miR-3673 891 1912 hsa-miR-367-3p 892 1913 hsa-miR-3674 893 1914hsa-miR-3675-3p 894 1915 hsa-miR-3675-5p 895 1916 hsa-miR-367-5p 8961917 hsa-miR-3676-3p 897 1918 hsa-miR-3676-5p 898 1919 hsa-miR-3677-3p899 1920 hsa-miR-3677-5p 900 1921 hsa-miR-3678-3p 901 1922hsa-miR-3678-5p 902 1923 hsa-miR-3679-3p 903 1924 hsa-miR-3679-5p 9041925 hsa-miR-3680-3p 905 1926 hsa-miR-3680-5p 906 1927 hsa-miR-3681-3p907 1928 hsa-miR-3681-5p 908 1929 hsa-miR-3682-3p 909 1930hsa-miR-3682-5p 910 1931 hsa-miR-3683 911 1932 hsa-miR-3684 912 1933hsa-miR-3685 913 1934 hsa-miR-3686 914 1935 hsa-miR-3687 915 1936hsa-miR-3688-3p 916 1937 hsa-miR-3688-5p 917 1938 hsa-miR-3689a-3p 9181939 hsa-miR-3689a-5p 919 1940 hsa-miR-3689b-3p 920 1941hsa-miR-3689b-5p 921 1942 hsa-miR-3689c 922 1943 hsa-miR-3689d 923 1944hsa-miR-3689e 924 1945 hsa-miR-3689f 925 1946 hsa-miR-3690 926 1947hsa-miR-3691-3p 927 1948 hsa-miR-3691-5p 928 1949 hsa-miR-3692-3p 9291950 hsa-miR-3692-5p 930 1951 hsa-miR-369-3p 931 1952 hsa-miR-369-5p 9321953 hsa-miR-370 933 1954 hsa-miR-3713 934 1955 hsa-miR-3714 935 1956hsa-miR-371a-3p 936 1957 hsa-miR-371a-5p 937 1958 hsa-miR-371b-3p 9381959 hsa-miR-371b-5p 939 1960 hsa-miR-372 940 1961 hsa-miR-373-3p 9411962 hsa-miR-373-5p 942 1963 hsa-miR-374a-3p 943 1964 hsa-miR-374a-5p944 1965 hsa-miR-374b-3p 945 1966 hsa-miR-374b-5p 946 1967hsa-miR-374c-3p 947 1968 hsa-miR-374c-5p 948 1969 hsa-miR-375 949 1970hsa-miR-376a-2-5p 950 1971 hsa-miR-376a-3p 951 1972 hsa-miR-376a-5p 9521973 hsa-miR-376b-3p 953 1974 hsa-miR-376b-5p 954 1975 hsa-miR-376c-3p955 1976 hsa-miR-376c-5p 956 1977 hsa-miR-377-3p 957 1978 hsa-miR-377-5p958 1979 hsa-miR-378a-3p 959 1980 hsa-miR-378a-5p 960 1981 hsa-miR-378b961 1982 hsa-miR-378c 962 1983 hsa-miR-378d 963 1984 hsa-miR-378e 9641985 hsa-miR-378f 965 1986 hsa-miR-378g 966 1987 hsa-miR-378h 967 1988hsa-miR-378i 968 1989 hsa-miR-378j 969 1990 hsa-miR-379-3p 970 1991hsa-miR-379-5p 971 1992 hsa-miR-380-3p 972 1993 hsa-miR-380-5p 973 1994hsa-miR-381-3p 974 1995 hsa-miR-381-5p 975 1996 hsa-miR-382-3p 976 1997hsa-miR-382-5p 977 1998 hsa-miR-383 978 1999 hsa-miR-384 979 2000hsa-miR-3907 980 2001 hsa-miR-3908 981 2002 hsa-miR-3909 982 2003hsa-miR-3910 983 2004 hsa-miR-3911 984 2005 hsa-miR-3912 985 2006hsa-miR-3913-3p 986 2007 hsa-miR-3913-5p 987 2008 hsa-miR-3914 988 2009hsa-miR-3915 989 2010 hsa-miR-3916 990 2011 hsa-miR-3917 991 2012hsa-miR-3918 992 2013 hsa-miR-3919 993 2014 hsa-miR-3920 994 2015hsa-miR-3921 995 2016 hsa-miR-3922-3p 996 2017 hsa-miR-3922-5p 997 2018hsa-miR-3923 998 2019 hsa-miR-3924 999 2020 hsa-miR-3925-3p 1000 2021hsa-miR-3925-5p 1001 2022 hsa-miR-3926 1002 2023 hsa-miR-3927-3p 10032024 hsa-miR-3927-5p 1004 2025 hsa-miR-3928 1005 2026 hsa-miR-3929 10062027 hsa-miR-3934-3p 1007 2028 hsa-miR-3934-5p 1008 2029 hsa-miR-39351009 2030 hsa-miR-3936 1010 2031 hsa-miR-3937 1011 2032 hsa-miR-39381012 2033 hsa-miR-3939 1013 2034 hsa-miR-3940-3p 1014 2035hsa-miR-3940-5p 1015 2036 hsa-miR-3941 1016 2037 hsa-miR-3942-3p 10172038 hsa-miR-3942-5p 1018 2039 hsa-miR-3943 1019 2040 hsa-miR-3944-3p1020 2041 hsa-miR-3944-5p 1021 2042 hsa-miR-3945 1022 2043 hsa-miR-39601023 2044 hsa-miR-3972 1024 2045 hsa-miR-3973 1025 2046 hsa-miR-39741026 2047 hsa-miR-3975 1027 2048 hsa-miR-3976 1028 2049 hsa-miR-39771029 2050 hsa-miR-3978 1030 2051 hsa-miR-409-3p 1031 2052 hsa-miR-409-5p1032 2053 hsa-miR-410 1033 2054 hsa-miR-411-3p 1034 2055 hsa-miR-411-5p1035 2056 hsa-miR-412 1036 2057 hsa-miR-421 1037 2058 hsa-miR-422a 10382059 hsa-miR-423-3p 1039 2060 hsa-miR-423-5p 1040 2061 hsa-miR-424-3p1041 2062 hsa-miR-424-5p 1042 2063 hsa-miR-4251 1043 2064 hsa-miR-42521044 2065 hsa-miR-4253 1045 2066 hsa-miR-425-3p 1046 2067 hsa-miR-42541047 2068 hsa-miR-4255 1048 2069 hsa-miR-425-5p 1049 2070 hsa-miR-42561050 2071 hsa-miR-4257 1051 2072 hsa-miR-4258 1052 2073 hsa-miR-42591053 2074 hsa-miR-4260 1054 2075 hsa-miR-4261 1055 2076 hsa-miR-42621056 2077 hsa-miR-4263 1057 2078 hsa-miR-4264 1058 2079 hsa-miR-42651059 2080 hsa-miR-4266 1060 2081 hsa-miR-4267 1061 2082 hsa-miR-42681062 2083 hsa-miR-4269 1063 2084 hsa-miR-4270 1064 2085 hsa-miR-42711065 2086 hsa-miR-4272 1066 2087 hsa-miR-4273 1067 2088 hsa-miR-42741068 2089 hsa-miR-4275 1069 2090 hsa-miR-4276 1070 2091 hsa-miR-42771071 2092 hsa-miR-4278 1072 2093 hsa-miR-4279 1073 2094 hsa-miR-42801074 2095 hsa-miR-4281 1075 2096 hsa-miR-4282 1076 2097 hsa-miR-42831077 2098 hsa-miR-4284 1078 2099 hsa-miR-4285 1079 2100 hsa-miR-42861080 2101 hsa-miR-4287 1081 2102 hsa-miR-4288 1082 2103 hsa-miR-42891083 2104 hsa-miR-429 1084 2105 hsa-miR-4290 1085 2106 hsa-miR-4291 10862107 hsa-miR-4292 1087 2108 hsa-miR-4293 1088 2109 hsa-miR-4294 10892110 hsa-miR-4295 1090 2111 hsa-miR-4296 1091 2112 hsa-miR-4297 10922113 hsa-miR-4298 1093 2114 hsa-miR-4299 1094 2115 hsa-miR-4300 10952116 hsa-miR-4301 1096 2117 hsa-miR-4302 1097 2118 hsa-miR-4303 10982119 hsa-miR-4304 1099 2120 hsa-miR-4305 1100 2121 hsa-miR-4306 11012122 hsa-miR-4307 1102 2123 hsa-miR-4308 1103 2124 hsa-miR-4309 11042125 hsa-miR-4310 1105 2126 hsa-miR-4311 1106 2127 hsa-miR-4312 11072128 hsa-miR-4313 1108 2129 hsa-miR-431-3p 1109 2130 hsa-miR-4314 11102131 hsa-miR-4315 1111 2132 hsa-miR-431-5p 1112 2133 hsa-miR-4316 11132134 hsa-miR-4317 1114 2135 hsa-miR-4318 1115 2136 hsa-miR-4319 11162137 hsa-miR-4320 1117 2138 hsa-miR-4321 1118 2139 hsa-miR-4322 11192140 hsa-miR-4323 1120 2141 hsa-miR-432-3p 1121 2142 hsa-miR-4324 11222143 hsa-miR-4325 1123 2144 hsa-miR-432-5p 1124 2145 hsa-miR-4326 11252146 hsa-miR-4327 1126 2147 hsa-miR-4328 1127 2148 hsa-miR-4329 11282149 hsa-miR-433 1129 2150 hsa-miR-4330 1130 2151 hsa-miR-4417 1131 2152hsa-miR-4418 1132 2153 hsa-miR-4419a 1133 2154 hsa-miR-4419b 1134 2155hsa-miR-4420 1135 2156 hsa-miR-4421 1136 2157 hsa-miR-4422 1137 2158hsa-miR-4423-3p 1138 2159 hsa-miR-4423-5p 1139 2160 hsa-miR-4424 11402161 hsa-miR-4425 1141 2162 hsa-miR-4426 1142 2163 hsa-miR-4427 11432164 hsa-miR-4428 1144 2165 hsa-miR-4429 1145 2166 hsa-miR-4430 11462167 hsa-miR-4431 1147 2168 hsa-miR-4432 1148 2169 hsa-miR-4433-3p 11492170 hsa-miR-4433-5p 1150 2171 hsa-miR-4434 1151 2172 hsa-miR-4435 11522173 hsa-miR-4436a 1153 2174 hsa-miR-4436b-3p 1154 2175 hsa-miR-4436b-5p1155 2176 hsa-miR-4437 1156 2177 hsa-miR-4438 1157 2178 hsa-miR-44391158 2179 hsa-miR-4440 1159 2180 hsa-miR-4441 1160 2181 hsa-miR-44421161 2182 hsa-miR-4443 1162 2183 hsa-miR-4444 1163 2184 hsa-miR-4445-3p1164 2185 hsa-miR-4445-5p 1165 2186 hsa-miR-4446-3p 1166 2187hsa-miR-4446-5p 1167 2188 hsa-miR-4447 1168 2189 hsa-miR-4448 1169 2190hsa-miR-4449 1170 2191 hsa-miR-4450 1171 2192 hsa-miR-4451 1172 2193hsa-miR-4452 1173 2194 hsa-miR-4453 1174 2195 hsa-miR-4454 1175 2196hsa-miR-4455 1176 2197 hsa-miR-4456 1177 2198 hsa-miR-4457 1178 2199hsa-miR-4458 1179 2200 hsa-miR-4459 1180 2201 hsa-miR-4460 1181 2202hsa-miR-4461 1182 2203 hsa-miR-4462 1183 2204 hsa-miR-4463 1184 2205hsa-miR-4464 1185 2206 hsa-miR-4465 1186 2207 hsa-miR-4466 1187 2208hsa-miR-4467 1188 2209 hsa-miR-4468 1189 2210 hsa-miR-4469 1190 2211hsa-miR-4470 1191 2212 hsa-miR-4471 2213 3234 hsa-miR-4472 2214 3235hsa-miR-4473 2215 3236 hsa-miR-4474-3p 2216 3237 hsa-miR-4474-5p 22173238 hsa-miR-4475 2218 3239 hsa-miR-4476 2219 3240 hsa-miR-4477a 22203241 hsa-miR-4477b 2221 3242 hsa-miR-4478 2222 3243 hsa-miR-4479 22233244 hsa-miR-448 2224 3245 hsa-miR-4480 2225 3246 hsa-miR-4481 2226 3247hsa-miR-4482-3p 2227 3248 hsa-miR-4482-5p 2228 3249 hsa-miR-4483 22293250 hsa-miR-4484 2230 3251 hsa-miR-4485 2231 3252 hsa-miR-4486 22323253 hsa-miR-4487 2233 3254 hsa-miR-4488 2234 3255 hsa-miR-4489 22353256 hsa-miR-4490 2236 3257 hsa-miR-4491 2237 3258 hsa-miR-4492 22383259 hsa-miR-4493 2239 3260 hsa-miR-4494 2240 3261 hsa-miR-4495 22413262 hsa-miR-4496 2242 3263 hsa-miR-4497 2243 3264 hsa-miR-4498 22443265 hsa-miR-4499 2245 3266 hsa-miR-449a 2246 3267 hsa-miR-449b-3p 22473268 hsa-miR-449b-5p 2248 3269 hsa-miR-449c-3p 2249 3270 hsa-miR-449c-5p2250 3271 hsa-miR-4500 2251 3272 hsa-miR-4501 2252 3273 hsa-miR-45022253 3274 hsa-miR-4503 2254 3275 hsa-miR-4504 2255 3276 hsa-miR-45052256 3277 hsa-miR-4506 2257 3278 hsa-miR-4507 2258 3279 hsa-miR-45082259 3280 hsa-miR-4509 2260 3281 hsa-miR-450a-3p 2261 3282hsa-miR-450a-5p 2262 3283 hsa-miR-450b-3p 2263 3284 hsa-miR-450b-5p 22643285 hsa-miR-4510 2265 3286 hsa-miR-4511 2266 3287 hsa-miR-4512 22673288 hsa-miR-4513 2268 3289 hsa-miR-4514 2269 3290 hsa-miR-4515 22703291 hsa-miR-4516 2271 3292 hsa-miR-4517 2272 3293 hsa-miR-4518 22733294 hsa-miR-4519 2274 3295 hsa-miR-451a 2275 3296 hsa-miR-451b 22763297 hsa-miR-4520a-3p 2277 3298 hsa-miR-4520a-5p 2278 3299hsa-miR-4520b-3p 2279 3300 hsa-miR-4520b-5p 2280 3301 hsa-miR-4521 22813302 hsa-miR-4522 2282 3303 hsa-miR-4523 2283 3304 hsa-miR-452-3p 22843305 hsa-miR-4524a-3p 2285 3306 hsa-miR-4524a-5p 2286 3307hsa-miR-4524b-3p 2287 3308 hsa-miR-4524b-5p 2288 3309 hsa-miR-4525 22893310 hsa-miR-452-5p 2290 3311 hsa-miR-4526 2291 3312 hsa-miR-4527 22923313 hsa-miR-4528 2293 3314 hsa-miR-4529-3p 2294 3315 hsa-miR-4529-5p2295 3316 hsa-miR-4530 2296 3317 hsa-miR-4531 2297 3318 hsa-miR-45322298 3319 hsa-miR-4533 2299 3320 hsa-miR-4534 2300 3321 hsa-miR-45352301 3322 hsa-miR-4536-3p 2302 3323 hsa-miR-4536-5p 2303 3324hsa-miR-4537 2304 3325 hsa-miR-4538 2305 3326 hsa-miR-4539 2306 3327hsa-miR-4540 2307 3328 hsa-miR-454-3p 2308 3329 hsa-miR-454-5p 2309 3330hsa-miR-455-3p 2310 3331 hsa-miR-455-5p 2311 3332 hsa-miR-4632-3p 23123333 hsa-miR-4632-5p 2313 3334 hsa-miR-4633-3p 2314 3335 hsa-miR-4633-5p2315 3336 hsa-miR-4634 2316 3337 hsa-miR-4635 2317 3338 hsa-miR-46362318 3339 hsa-miR-4637 2319 3340 hsa-miR-4638-3p 2320 3341hsa-miR-4638-5p 2321 3342 hsa-miR-4639-3p 2322 3343 hsa-miR-4639-5p 23233344 hsa-miR-4640-3p 2324 3345 hsa-miR-4640-5p 2325 3346 hsa-miR-46412326 3347 hsa-miR-4642 2327 3348 hsa-miR-4643 2328 3349 hsa-miR-46442329 3350 hsa-miR-4645-3p 2330 3351 hsa-miR-4645-5p 2331 3352hsa-miR-4646-3p 2332 3353 hsa-miR-4646-5p 2333 3354 hsa-miR-4647 23343355 hsa-miR-4648 2335 3356 hsa-miR-4649-3p 2336 3357 hsa-miR-4649-5p2337 3358 hsa-miR-4650-3p 2338 3359 hsa-miR-4650-5p 2339 3360hsa-miR-4651 2340 3361 hsa-miR-4652-3p 2341 3362 hsa-miR-4652-5p 23423363 hsa-miR-4653-3p 2343 3364 hsa-miR-4653-5p 2344 3365 hsa-miR-46542345 3366 hsa-miR-4655-3p 2346 3367 hsa-miR-4655-5p 2347 3368hsa-miR-4656 2348 3369 hsa-miR-4657 2349 3370 hsa-miR-4658 2350 3371hsa-miR-4659a-3p 2351 3372 hsa-miR-4659a-5p 2352 3373 hsa-miR-4659b-3p2353 3374 hsa-miR-4659b-5p 2354 3375 hsa-miR-466 2355 3376 hsa-miR-46602356 3377 hsa-miR-4661-3p 2357 3378 hsa-miR-4661-5p 2358 3379hsa-miR-4662a-3p 2359 3380 hsa-miR-4662a-5p 2360 3381 hsa-miR-4662b 23613382 hsa-miR-4663 2362 3383 hsa-miR-4664-3p 2363 3384 hsa-miR-4664-5p2364 3385 hsa-miR-4665-3p 2365 3386 hsa-miR-4665-5p 2366 3387hsa-miR-4666a-3p 2367 3388 hsa-miR-4666a-5p 2368 3389 hsa-miR-4666b 23693390 hsa-miR-4667-3p 2370 3391 hsa-miR-4667-5p 2371 3392 hsa-miR-4668-3p2372 3393 hsa-miR-4668-5p 2373 3394 hsa-miR-4669 2374 3395hsa-miR-4670-3p 2375 3396 hsa-miR-4670-5p 2376 3397 hsa-miR-4671-3p 23773398 hsa-miR-4671-5p 2378 3399 hsa-miR-4672 2379 3400 hsa-miR-4673 23803401 hsa-miR-4674 2381 3402 hsa-miR-4675 2382 3403 hsa-miR-4676-3p 23833404 hsa-miR-4676-5p 2384 3405 hsa-miR-4677-3p 2385 3406 hsa-miR-4677-5p2386 3407 hsa-miR-4678 2387 3408 hsa-miR-4679 2388 3409 hsa-miR-4680-3p2389 3410 hsa-miR-4680-5p 2390 3411 hsa-miR-4681 2391 3412 hsa-miR-46822392 3413 hsa-miR-4683 2393 3414 hsa-miR-4684-3p 2394 3415hsa-miR-4684-5p 2395 3416 hsa-miR-4685-3p 2396 3417 hsa-miR-4685-5p 23973418 hsa-miR-4686 2398 3419 hsa-miR-4687-3p 2399 3420 hsa-miR-4687-5p2400 3421 hsa-miR-4688 2401 3422 hsa-miR-4689 2402 3423 hsa-miR-4690-3p2403 3424 hsa-miR-4690-5p 2404 3425 hsa-miR-4691-3p 2405 3426hsa-miR-4691-5p 2406 3427 hsa-miR-4692 2407 3428 hsa-miR-4693-3p 24083429 hsa-miR-4693-5p 2409 3430 hsa-miR-4694-3p 2410 3431 hsa-miR-4694-5p2411 3432 hsa-miR-4695-3p 2412 3433 hsa-miR-4695-5p 2413 3434hsa-miR-4696 2414 3435 hsa-miR-4697-3p 2415 3436 hsa-miR-4697-5p 24163437 hsa-miR-4698 2417 3438 hsa-miR-4699-3p 2418 3439 hsa-miR-4699-5p2419 3440 hsa-miR-4700-3p 2420 3441 hsa-miR-4700-5p 2421 3442hsa-miR-4701-3p 2422 3443 hsa-miR-4701-5p 2423 3444 hsa-miR-4703-3p 24243445 hsa-miR-4703-5p 2425 3446 hsa-miR-4704-3p 2426 3447 hsa-miR-4704-5p2427 3448 hsa-miR-4705 2428 3449 hsa-miR-4706 2429 3450 hsa-miR-4707-3p2430 3451 hsa-miR-4707-5p 2431 3452 hsa-miR-4708-3p 2432 3453hsa-miR-4708-5p 2433 3454 hsa-miR-4709-3p 2434 3455 hsa-miR-4709-5p 24353456 hsa-miR-4710 2436 3457 hsa-miR-4711-3p 2437 3458 hsa-miR-4711-5p2438 3459 hsa-miR-4712-3p 2439 3460 hsa-miR-4712-5p 2440 3461hsa-miR-4713-3p 2441 3462 hsa-miR-4713-5p 2442 3463 hsa-miR-4714-3p 24433464 hsa-miR-4714-5p 2444 3465 hsa-miR-4715-3p 2445 3466 hsa-miR-4715-5p2446 3467 hsa-miR-4716-3p 2447 3468 hsa-miR-4716-5p 2448 3469hsa-miR-4717-3p 2449 3470 hsa-miR-4717-5p 2450 3471 hsa-miR-4718 24513472 hsa-miR-4719 2452 3473 hsa-miR-4720-3p 2453 3474 hsa-miR-4720-5p2454 3475 hsa-miR-4721 2455 3476 hsa-miR-4722-3p 2456 3477hsa-miR-4722-5p 2457 3478 hsa-miR-4723-3p 2458 3479 hsa-miR-4723-5p 24593480 hsa-miR-4724-3p 2460 3481 hsa-miR-4724-5p 2461 3482 hsa-miR-4725-3p2462 3483 hsa-miR-4725-5p 2463 3484 hsa-miR-4726-3p 2464 3485hsa-miR-4726-5p 2465 3486 hsa-miR-4727-3p 2466 3487 hsa-miR-4727-5p 24673488 hsa-miR-4728-3p 2468 3489 hsa-miR-4728-5p 2469 3490 hsa-miR-47292470 3491 hsa-miR-4730 2471 3492 hsa-miR-4731-3p 2472 3493hsa-miR-4731-5p 2473 3494 hsa-miR-4732-3p 2474 3495 hsa-miR-4732-5p 24753496 hsa-miR-4733-3p 2476 3497 hsa-miR-4733-5p 2477 3498 hsa-miR-47342478 3499 hsa-miR-4735-3p 2479 3500 hsa-miR-4735-5p 2480 3501hsa-miR-4736 2481 3502 hsa-miR-4737 2482 3503 hsa-miR-4738-3p 2483 3504hsa-miR-4738-5p 2484 3505 hsa-miR-4739 2485 3506 hsa-miR-4740-3p 24863507 hsa-miR-4740-5p 2487 3508 hsa-miR-4741 2488 3509 hsa-miR-4742-3p2489 3510 hsa-miR-4742-5p 2490 3511 hsa-miR-4743-3p 2491 3512hsa-miR-4743-5p 2492 3513 hsa-miR-4744 2493 3514 hsa-miR-4745-3p 24943515 hsa-miR-4745-5p 2495 3516 hsa-miR-4746-3p 2496 3517 hsa-miR-4746-5p2497 3518 hsa-miR-4747-3p 2498 3519 hsa-miR-4747-5p 2499 3520hsa-miR-4748 2500 3521 hsa-miR-4749-3p 2501 3522 hsa-miR-4749-5p 25023523 hsa-miR-4750-3p 2503 3524 hsa-miR-4750-5p 2504 3525 hsa-miR-47512505 3526 hsa-miR-4752 2506 3527 hsa-miR-4753-3p 2507 3528hsa-miR-4753-5p 2508 3529 hsa-miR-4754 2509 3530 hsa-miR-4755-3p 25103531 hsa-miR-4755-5p 2511 3532 hsa-miR-4756-3p 2512 3533 hsa-miR-4756-5p2513 3534 hsa-miR-4757-3p 2514 3535 hsa-miR-4757-5p 2515 3536hsa-miR-4758-3p 2516 3537 hsa-miR-4758-5p 2517 3538 hsa-miR-4759 25183539 hsa-miR-4760-3p 2519 3540 hsa-miR-4760-5p 2520 3541 hsa-miR-4761-3p2521 3542 hsa-miR-4761-5p 2522 3543 hsa-miR-4762-3p 2523 3544hsa-miR-4762-5p 2524 3545 hsa-miR-4763-3p 2525 3546 hsa-miR-4763-5p 25263547 hsa-miR-4764-3p 2527 3548 hsa-miR-4764-5p 2528 3549 hsa-miR-47652529 3550 hsa-miR-4766-3p 2530 3551 hsa-miR-4766-5p 2531 3552hsa-miR-4767 2532 3553 hsa-miR-4768-3p 2533 3554 hsa-miR-4768-5p 25343555 hsa-miR-4769-3p 2535 3556 hsa-miR-4769-5p 2536 3557 hsa-miR-47702537 3558 hsa-miR-4771 2538 3559 hsa-miR-4772-3p 2539 3560hsa-miR-4772-5p 2540 3561 hsa-miR-4773 2541 3562 hsa-miR-4774-3p 25423563 hsa-miR-4774-5p 2543 3564 hsa-miR-4775 2544 3565 hsa-miR-4776-3p2545 3566 hsa-miR-4776-5p 2546 3567 hsa-miR-4777-3p 2547 3568hsa-miR-4777-5p 2548 3569 hsa-miR-4778-3p 2549 3570 hsa-miR-4778-5p 25503571 hsa-miR-4779 2551 3572 hsa-miR-4780 2552 3573 hsa-miR-4781-3p 25533574 hsa-miR-4781-5p 2554 3575 hsa-miR-4782-3p 2555 3576 hsa-miR-4782-5p2556 3577 hsa-miR-4783-3p 2557 3578 hsa-miR-4783-5p 2558 3579hsa-miR-4784 2559 3580 hsa-miR-4785 2560 3581 hsa-miR-4786-3p 2561 3582hsa-miR-4786-5p 2562 3583 hsa-miR-4787-3p 2563 3584 hsa-miR-4787-5p 25643585 hsa-miR-4788 2565 3586 hsa-miR-4789-3p 2566 3587 hsa-miR-4789-5p2567 3588 hsa-miR-4790-3p 2568 3589 hsa-miR-4790-5p 2569 3590hsa-miR-4791 2570 3591 hsa-miR-4792 2571 3592 hsa-miR-4793-3p 2572 3593hsa-miR-4793-5p 2573 3594 hsa-miR-4794 2574 3595 hsa-miR-4795-3p 25753596 hsa-miR-4795-5p 2576 3597 hsa-miR-4796-3p 2577 3598 hsa-miR-4796-5p2578 3599 hsa-miR-4797-3p 2579 3600 hsa-miR-4797-5p 2580 3601hsa-miR-4798-3p 2581 3602 hsa-miR-4798-5p 2582 3603 hsa-miR-4799-3p 25833604 hsa-miR-4799-5p 2584 3605 hsa-miR-4800-3p 2585 3606 hsa-miR-4800-5p2586 3607 hsa-miR-4801 2587 3608 hsa-miR-4802-3p 2588 3609hsa-miR-4802-5p 2589 3610 hsa-miR-4803 2590 3611 hsa-miR-4804-3p 25913612 hsa-miR-4804-5p 2592 3613 hsa-miR-483-3p 2593 3614 hsa-miR-483-5p2594 3615 hsa-miR-484 2595 3616 hsa-miR-485-3p 2596 3617 hsa-miR-485-5p2597 3618 hsa-miR-486-3p 2598 3619 hsa-miR-486-5p 2599 3620 hsa-miR-487a2600 3621 hsa-miR-487b 2601 3622 hsa-miR-488-3p 2602 3623 hsa-miR-488-5p2603 3624 hsa-miR-489 2604 3625 hsa-miR-490-3p 2605 3626 hsa-miR-490-5p2606 3627 hsa-miR-491-3p 2607 3628 hsa-miR-491-5p 2608 3629 hsa-miR-4922609 3630 hsa-miR-493-3p 2610 3631 hsa-miR-493-5p 2611 3632 hsa-miR-4942612 3633 hsa-miR-495-3p 2613 3634 hsa-miR-495-5p 2614 3635 hsa-miR-4962615 3636 hsa-miR-497-3p 2616 3637 hsa-miR-497-5p 2617 3638 hsa-miR-4982618 3639 hsa-miR-4999-3p 2619 3640 hsa-miR-4999-5p 2620 3641hsa-miR-499a-3p 2621 3642 hsa-miR-499a-5p 2622 3643 hsa-miR-499b-3p 26233644 hsa-miR-499b-5p 2624 3645 hsa-miR-5000-3p 2625 3646 hsa-miR-5000-5p2626 3647 hsa-miR-5001-3p 2627 3648 hsa-miR-5001-5p 2628 3649hsa-miR-5002-3p 2629 3650 hsa-miR-5002-5p 2630 3651 hsa-miR-5003-3p 26313652 hsa-miR-5003-5p 2632 3653 hsa-miR-5004-3p 2633 3654 hsa-miR-5004-5p2634 3655 hsa-miR-5006-3p 2635 3656 hsa-miR-5006-5p 2636 3657hsa-miR-5007-3p 2637 3658 hsa-miR-5007-5p 2638 3659 hsa-miR-5008-3p 26393660 hsa-miR-5008-5p 2640 3661 hsa-miR-5009-3p 2641 3662 hsa-miR-5009-5p2642 3663 hsa-miR-500a-3p 2643 3664 hsa-miR-500a-5p 2644 3665hsa-miR-500b 2645 3666 hsa-miR-5010-3p 2646 3667 hsa-miR-5010-5p 26473668 hsa-miR-5011-3p 2648 3669 hsa-miR-5011-5p 2649 3670 hsa-miR-501-3p2650 3671 hsa-miR-501-5p 2651 3672 hsa-miR-502-3p 2652 3673hsa-miR-502-5p 2653 3674 hsa-miR-503-3p 2654 3675 hsa-miR-503-5p 26553676 hsa-miR-504 2656 3677 hsa-miR-5047 2657 3678 hsa-miR-505-3p 26583679 hsa-miR-505-5p 2659 3680 hsa-miR-506-3p 2660 3681 hsa-miR-506-5p2661 3682 hsa-miR-507 2662 3683 hsa-miR-508-3p 2663 3684 hsa-miR-508-5p2664 3685 hsa-miR-5087 2665 3686 hsa-miR-5088 2666 3687 hsa-miR-5089-3p2667 3688 hsa-miR-5089-5p 2668 3689 hsa-miR-5090 2669 3690 hsa-miR-50912670 3691 hsa-miR-5092 2671 3692 hsa-miR-5093 2672 3693 hsa-miR-509-3-5p2673 3694 hsa-miR-509-3p 2674 3695 hsa-miR-5094 2675 3696 hsa-miR-50952676 3697 hsa-miR-509-5p 2677 3698 hsa-miR-5096 2678 3699 hsa-miR-5102679 3700 hsa-miR-5100 2680 3701 hsa-miR-511 2681 3702 hsa-miR-512-3p2682 3703 hsa-miR-512-5p 2683 3704 hsa-miR-513a-3p 2684 3705hsa-miR-513a-5p 2685 3706 hsa-miR-513b 2686 3707 hsa-miR-513c-3p 26873708 hsa-miR-513c-5p 2688 3709 hsa-miR-514a-3p 2689 3710 hsa-miR-514a-5p2690 3711 hsa-miR-514b-3p 2691 3712 hsa-miR-514b-5p 2692 3713hsa-miR-515-3p 2693 3714 hsa-miR-515-5p 2694 3715 hsa-miR-516a-3p 26953716 hsa-miR-516a-5p 2696 3717 hsa-miR-516b-3p 2697 3718 hsa-miR-516b-5p2698 3719 hsa-miR-517-5p 2699 3720 hsa-miR-517a-3p 2700 3721hsa-miR-517b-3p 2701 3722 hsa-miR-517c-3p 2702 3723 hsa-miR-5186 27033724 hsa-miR-5187-3p 2704 3725 hsa-miR-5187-5p 2705 3726 hsa-miR-51882706 3727 hsa-miR-5189 2707 3728 hsa-miR-518a-3p 2708 3729hsa-miR-518a-5p 2709 3730 hsa-miR-518b 2710 3731 hsa-miR-518c-3p 27113732 hsa-miR-518c-5p 2712 3733 hsa-miR-518d-3p 2713 3734 hsa-miR-518d-5p2714 3735 hsa-miR-518e-3p 2715 3736 hsa-miR-518e-5p 2716 3737hsa-miR-518f-3p 2717 3738 hsa-miR-518f-5p 2718 3739 hsa-miR-5190 27193740 hsa-miR-5191 2720 3741 hsa-miR-5192 2721 3742 hsa-miR-5193 27223743 hsa-miR-5194 2723 3744 hsa-miR-5195-3p 2724 3745 hsa-miR-5195-5p2725 3746 hsa-miR-5196-3p 2726 3747 hsa-miR-5196-5p 2727 3748hsa-miR-5197-3p 2728 3749 hsa-miR-5197-5p 2729 3750 hsa-miR-519a-3p 27303751 hsa-miR-519a-5p 2731 3752 hsa-miR-519b-3p 2732 3753 hsa-miR-519b-5p2733 3754 hsa-miR-519c-3p 2734 3755 hsa-miR-519c-5p 2735 3756hsa-miR-519d 2736 3757 hsa-miR-519e-3p 2737 3758 hsa-miR-519e-5p 27383759 hsa-miR-520a-3p 2739 3760 hsa-miR-520a-5p 2740 3761 hsa-miR-520b2741 3762 hsa-miR-520c-3p 2742 3763 hsa-miR-520c-5p 2743 3764hsa-miR-520d-3p 2744 3765 hsa-miR-520d-5p 2745 3766 hsa-miR-520e 27463767 hsa-miR-520f 2747 3768 hsa-miR-520g 2748 3769 hsa-miR-520h 27493770 hsa-miR-521 2750 3771 hsa-miR-522-3p 2751 3772 hsa-miR-522-5p 27523773 hsa-miR-523-3p 2753 3774 hsa-miR-523-5p 2754 3775 hsa-miR-524-3p2755 3776 hsa-miR-524-5p 2756 3777 hsa-miR-525-3p 2757 3778hsa-miR-525-5p 2758 3779 hsa-miR-526a 2759 3780 hsa-miR-526b-3p 27603781 hsa-miR-526b-5p 2761 3782 hsa-miR-527 2762 3783 hsa-miR-532-3p 27633784 hsa-miR-532-5p 2764 3785 hsa-miR-539-3p 2765 3786 hsa-miR-539-5p2766 3787 hsa-miR-541-3p 2767 3788 hsa-miR-541-5p 2768 3789hsa-miR-542-3p 2769 3790 hsa-miR-542-5p 2770 3791 hsa-miR-543 2771 3792hsa-miR-544a 2772 3793 hsa-miR-544b 2773 3794 hsa-miR-545-3p 2774 3795hsa-miR-545-5p 2775 3796 hsa-miR-548 2776 3797 hsa-miR-548-3p 2777 3798hsa-miR-548-5p 2778 3799 hsa-miR-548a 2779 3800 hsa-miR-548a-3p 27803801 hsa-miR-548a-5p 2781 3802 hsa-miR-548aa 2782 3803 hsa-miR-548ab2783 3804 hsa-miR-548ac 2784 3805 hsa-miR-548ad 2785 3806 hsa-miR-548ae2786 3807 hsa-miR-548ag 2787 3808 hsa-miR-548ah-3p 2788 3809hsa-miR-548ah-5p 2789 3810 hsa-miR-548ai 2790 3811 hsa-miR-548aj-3p 27913812 hsa-miR-548aj-5p 2792 3813 hsa-miR-548ak 2793 3814 hsa-miR-548al2794 3815 hsa-miR-548am-3p 2795 3816 hsa-miR-548am-5p 2796 3817hsa-miR-548an 2797 3818 hsa-miR-548ao-3p 2798 3819 hsa-miR-548ao-5p 27993820 hsa-miR-548ap-3p 2800 3821 hsa-miR-548ap-5p 2801 3822hsa-miR-548aq-3p 2802 3823 hsa-miR-548aq-5p 2803 3824 hsa-miR-548ar-3p2804 3825 hsa-miR-548ar-5p 2805 3826 hsa-miR-548as-3p 2806 3827hsa-miR-548as-5p 2807 3828 hsa-miR-548at-3p 2808 3829 hsa-miR-548at-5p2809 3830 hsa-miR-548au-3p 2810 3831 hsa-miR-548au-5p 2811 3832hsa-miR-548av-3p 2812 3833 hsa-miR-548av-5p 2813 3834 hsa-miR-548aw 28143835 hsa-miR-548ay-3p 2815 3836 hsa-miR-548ay-5p 2816 3837hsa-miR-548az-3p 2817 3838 hsa-miR-548az-5p 2818 3839 hsa-miR-548b-3p2819 3840 hsa-miR-548b-5p 2820 3841 hsa-miR-548c-3p 2821 3842hsa-miR-548c-5p 2822 3843 hsa-miR-548d-3p 2823 3844 hsa-miR-548d-5p 28243845 hsa-miR-548e 2825 3846 hsa-miR-548f 2826 3847 hsa-miR-548g-3p 28273848 hsa-miR-548g-5p 2828 3849 hsa-miR-548h-3p 2829 3850 hsa-miR-548h-5p2830 3851 hsa-miR-548i 2831 3852 hsa-miR-548j 2832 3853 hsa-miR-548k2833 3854 hsa-miR-548l 2834 3855 hsa-miR-548m 2835 3856 hsa-miR-548n2836 3857 hsa-miR-548o-3p 2837 3858 hsa-miR-548o-5p 2838 3859hsa-miR-548p 2839 3860 hsa-miR-548q 2840 3861 hsa-miR-548s 2841 3862hsa-miR-548t-3p 2842 3863 hsa-miR-548t-5p 2843 3864 hsa-miR-548u 28443865 hsa-miR-548w 2845 3866 hsa-miR-548y 2846 3867 hsa-miR-548z 28473868 hsa-miR-549a 2848 3869 hsa-miR-550a-3-5p 2849 3870 hsa-miR-550a-3p2850 3871 hsa-miR-550a-5p 2851 3872 hsa-miR-550b-2-5p 2852 3873hsa-miR-550b-3p 2853 3874 hsa-miR-551a 2854 3875 hsa-miR-551b-3p 28553876 hsa-miR-551b-5p 2856 3877 hsa-miR-552 2857 3878 hsa-miR-553 28583879 hsa-miR-554 2859 3880 hsa-miR-555 2860 3881 hsa-miR-556-3p 28613882 hsa-miR-556-5p 2862 3883 hsa-miR-557 2863 3884 hsa-miR-5571-3p 28643885 hsa-miR-5571-5p 2865 3886 hsa-miR-5572 2866 3887 hsa-miR-5579-3p2867 3888 hsa-miR-5579-5p 2868 3889 hsa-miR-558 2869 3890hsa-miR-5580-3p 2870 3891 hsa-miR-5580-5p 2871 3892 hsa-miR-5581-3p 28723893 hsa-miR-5581-5p 2873 3894 hsa-miR-5582-3p 2874 3895 hsa-miR-5582-5p2875 3896 hsa-miR-5583-3p 2876 3897 hsa-miR-5583-5p 2877 3898hsa-miR-5584-3p 2878 3899 hsa-miR-5584-5p 2879 3900 hsa-miR-5585-3p 28803901 hsa-miR-5585-5p 2881 3902 hsa-miR-5586-3p 2882 3903 hsa-miR-5586-5p2883 3904 hsa-miR-5587-3p 2884 3905 hsa-miR-5587-5p 2885 3906hsa-miR-5588-3p 2886 3907 hsa-miR-5588-5p 2887 3908 hsa-miR-5589-3p 28883909 hsa-miR-5589-5p 2889 3910 hsa-miR-559 2890 3911 hsa-miR-5590-3p2891 3912 hsa-miR-5590-5p 2892 3913 hsa-miR-5591-3p 2893 3914hsa-miR-5591-5p 2894 3915 hsa-miR-561-3p 2895 3916 hsa-miR-561-5p 28963917 hsa-miR-562 2897 3918 hsa-miR-563 2898 3919 hsa-miR-564 2899 3920hsa-miR-566 2900 3921 hsa-miR-567 2901 3922 hsa-miR-568 2902 3923hsa-miR-5680 2903 3924 hsa-miR-5681a 2904 3925 hsa-miR-5681b 2905 3926hsa-miR-5682 2906 3927 hsa-miR-5683 2907 3928 hsa-miR-5684 2908 3929hsa-miR-5685 2909 3930 hsa-miR-5686 2910 3931 hsa-miR-5687 2911 3932hsa-miR-5688 2912 3933 hsa-miR-5689 2913 3934 hsa-miR-569 2914 3935hsa-miR-5690 2915 3936 hsa-miR-5691 2916 3937 hsa-miR-5692a 2917 3938hsa-miR-5692b 2918 3939 hsa-miR-5692c 2919 3940 hsa-miR-5693 2920 3941hsa-miR-5694 2921 3942 hsa-miR-5695 2922 3943 hsa-miR-5696 2923 3944hsa-miR-5697 2924 3945 hsa-miR-5698 2925 3946 hsa-miR-5699 2926 3947hsa-miR-5700 2927 3948 hsa-miR-5701 2928 3949 hsa-miR-5702 2929 3950hsa-miR-5703 2930 3951 hsa-miR-570-3p 2931 3952 hsa-miR-5704 2932 3953hsa-miR-5705 2933 3954 hsa-miR-570-5p 2934 3955 hsa-miR-5706 2935 3956hsa-miR-5707 2936 3957 hsa-miR-5708 2937 3958 hsa-miR-571 2938 3959hsa-miR-572 2939 3960 hsa-miR-573 2940 3961 hsa-miR-5739 2941 3962hsa-miR-574-3p 2942 3963 hsa-miR-574-5p 2943 3964 hsa-miR-575 2944 3965hsa-miR-576-3p 2945 3966 hsa-miR-576-5p 2946 3967 hsa-miR-577 2947 3968hsa-miR-578 2948 3969 hsa-miR-5787 2949 3970 hsa-miR-579 2950 3971hsa-miR-580 2951 3972 hsa-miR-581 2952 3973 hsa-miR-582-3p 2953 3974hsa-miR-582-5p 2954 3975 hsa-miR-583 2955 3976 hsa-miR-584-3p 2956 3977hsa-miR-584-5p 2957 3978 hsa-miR-585 2958 3979 hsa-miR-586 2959 3980hsa-miR-587 2960 3981 hsa-miR-588 2961 3982 hsa-miR-589-3p 2962 3983hsa-miR-589-5p 2963 3984 hsa-miR-590-3p 2964 3985 hsa-miR-590-5p 29653986 hsa-miR-591 2966 3987 hsa-miR-592 2967 3988 hsa-miR-593-3p 29683989 hsa-miR-593-5p 2969 3990 hsa-miR-595 2970 3991 hsa-miR-596 29713992 hsa-miR-597 2972 3993 hsa-miR-598 2973 3994 hsa-miR-599 2974 3995hsa-miR-600 2975 3996 hsa-miR-601 2976 3997 hsa-miR-602 2977 3998hsa-miR-603 2978 3999 hsa-miR-604 2979 4000 hsa-miR-605 2980 4001hsa-miR-606 2981 4002 hsa-miR-6068 2982 4003 hsa-miR-6069 2983 4004hsa-miR-607 2984 4005 hsa-miR-6070 2985 4006 hsa-miR-6071 2986 4007hsa-miR-6072 2987 4008 hsa-miR-6073 2988 4009 hsa-miR-6074 2989 4010hsa-miR-6075 2990 4011 hsa-miR-6076 2991 4012 hsa-miR-6077 2992 4013hsa-miR-6078 2993 4014 hsa-miR-6079 2994 4015 hsa-miR-608 2995 4016hsa-miR-6080 2996 4017 hsa-miR-6081 2997 4018 hsa-miR-6082 2998 4019hsa-miR-6083 2999 4020 hsa-miR-6084 3000 4021 hsa-miR-6085 3001 4022hsa-miR-6086 3002 4023 hsa-miR-6087 3003 4024 hsa-miR-6088 3004 4025hsa-miR-6089 3005 4026 hsa-miR-609 3006 4027 hsa-miR-6090 3007 4028hsa-miR-610 3008 4029 hsa-miR-611 3009 4030 hsa-miR-612 3010 4031hsa-miR-6124 3011 4032 hsa-miR-6125 3012 4033 hsa-miR-6126 3013 4034hsa-miR-6127 3014 4035 hsa-miR-6128 3015 4036 hsa-miR-6129 3016 4037hsa-miR-613 3017 4038 hsa-miR-6130 3018 4039 hsa-miR-6131 3019 4040hsa-miR-6132 3020 4041 hsa-miR-6133 3021 4042 hsa-miR-6134 3022 4043hsa-miR-614 3023 4044 hsa-miR-615-3p 3024 4045 hsa-miR-615-5p 3025 4046hsa-miR-616-3p 3026 4047 hsa-miR-6165 3027 4048 hsa-miR-616-5p 3028 4049hsa-miR-617 3029 4050 hsa-miR-618 3030 4051 hsa-miR-619 3031 4052hsa-miR-620 3032 4053 hsa-miR-621 3033 4054 hsa-miR-622 3034 4055hsa-miR-623 3035 4056 hsa-miR-624-3p 3036 4057 hsa-miR-624-5p 3037 4058hsa-miR-625-3p 3038 4059 hsa-miR-625-5p 3039 4060 hsa-miR-626 3040 4061hsa-miR-627 3041 4062 hsa-miR-628-3p 3042 4063 hsa-miR-628-5p 3043 4064hsa-miR-629-3p 3044 4065 hsa-miR-629-5p 3045 4066 hsa-miR-630 3046 4067hsa-miR-631 3047 4068 hsa-miR-632 3048 4069 hsa-miR-633 3049 4070hsa-miR-634 3050 4071 hsa-miR-635 3051 4072 hsa-miR-636 3052 4073hsa-miR-637 3053 4074 hsa-miR-638 3054 4075 hsa-miR-639 3055 4076hsa-miR-640 3056 4077 hsa-miR-641 3057 4078 hsa-miR-642a-3p 3058 4079hsa-miR-642a-5p 3059 4080 hsa-miR-642b-3p 3060 4081 hsa-miR-642b-5p 30614082 hsa-miR-643 3062 4083 hsa-miR-644a 3063 4084 hsa-miR-645 3064 4085hsa-miR-646 3065 4086 hsa-miR-647 3066 4087 hsa-miR-648 3067 4088hsa-miR-649 3068 4089 hsa-miR-6499-3p 3069 4090 hsa-miR-6499-5p 30704091 hsa-miR-650 3071 4092 hsa-miR-6500-3p 3072 4093 hsa-miR-6500-5p3073 4094 hsa-miR-6501-3p 3074 4095 hsa-miR-6501-5p 3075 4096hsa-miR-6502-3p 3076 4097 hsa-miR-6502-5p 3077 4098 hsa-miR-6503-3p 30784099 hsa-miR-6503-5p 3079 4100 hsa-miR-6504-3p 3080 4101 hsa-miR-6504-5p3081 4102 hsa-miR-6505-3p 3082 4103 hsa-miR-6505-5p 3083 4104hsa-miR-6506-3p 3084 4105 hsa-miR-6506-5p 3085 4106 hsa-miR-6507-3p 30864107 hsa-miR-6507-5p 3087 4108 hsa-miR-6508-3p 3088 4109 hsa-miR-6508-5p3089 4110 hsa-miR-6509-3p 3090 4111 hsa-miR-6509-5p 3091 4112hsa-miR-651 3092 4113 hsa-miR-6510-3p 3093 4114 hsa-miR-6510-5p 30944115 hsa-miR-6511a-3p 3095 4116 hsa-miR-6511a-5p 3096 4117hsa-miR-6511b-3p 3097 4118 hsa-miR-6511b-5p 3098 4119 hsa-miR-6512-3p3099 4120 hsa-miR-6512-5p 3100 4121 hsa-miR-6513-3p 3101 4122hsa-miR-6513-5p 3102 4123 hsa-miR-6514-3p 3103 4124 hsa-miR-6514-5p 31044125 hsa-miR-6515-3p 3105 4126 hsa-miR-6515-5p 3106 4127 hsa-miR-652-3p3107 4128 hsa-miR-652-5p 3108 4129 hsa-miR-653 3109 4130 hsa-miR-654-3p3110 4131 hsa-miR-654-5p 3111 4132 hsa-miR-655 3112 4133 hsa-miR-6563113 4134 hsa-miR-657 3114 4135 hsa-miR-658 3115 4136 hsa-miR-659-3p3116 4137 hsa-miR-659-5p 3117 4138 hsa-miR-660-3p 3118 4139hsa-miR-660-5p 3119 4140 hsa-miR-661 3120 4141 hsa-miR-662 3121 4142hsa-miR-663a 3122 4143 hsa-miR-663b 3123 4144 hsa-miR-664a-3p 3124 4145hsa-miR-664a-5p 3125 4146 hsa-miR-664b-3p 3126 4147 hsa-miR-664b-5p 31274148 hsa-miR-665 3128 4149 hsa-miR-668 3129 4150 hsa-miR-670 3130 4151hsa-miR-671-3p 3131 4152 hsa-miR-6715a-3p 3132 4153 hsa-miR-6715b-3p3133 4154 hsa-miR-6715b-5p 3134 4155 hsa-miR-671-5p 3135 4156hsa-miR-6716-3p 3136 4157 hsa-miR-6716-5p 3137 4158 hsa-miR-6717-5p 31384159 hsa-miR-6718-5p 3139 4160 hsa-miR-6719-3p 3140 4161 hsa-miR-6720-3p3141 4162 hsa-miR-6721-5p 3142 4163 hsa-miR-6722-3p 3143 4164hsa-miR-6722-5p 3144 4165 hsa-miR-6723-5p 3145 4166 hsa-miR-6724-5p 31464167 hsa-miR-675-3p 3147 4168 hsa-miR-675-5p 3148 4169 hsa-miR-676-3p3149 4170 hsa-miR-676-5p 3150 4171 hsa-miR-708-3p 3151 4172hsa-miR-708-5p 3152 4173 hsa-miR-711 3153 4174 hsa-miR-7-1-3p 3154 4175hsa-miR-718 3155 4176 hsa-miR-7-2-3p 3156 4177 hsa-miR-744-3p 3157 4178hsa-miR-744-5p 3158 4179 hsa-miR-758-3p 3159 4180 hsa-miR-758-5p 31604181 hsa-miR-759 3161 4182 hsa-miR-7-5p 3162 4183 hsa-miR-760 3163 4184hsa-miR-761 3164 4185 hsa-miR-762 3165 4186 hsa-miR-764 3166 4187hsa-miR-765 3167 4188 hsa-miR-766-3p 3168 4189 hsa-miR-766-5p 3169 4190hsa-miR-767-3p 3170 4191 hsa-miR-767-5p 3171 4192 hsa-miR-769-3p 31724193 hsa-miR-769-5p 3173 4194 hsa-miR-770-5p 3174 4195 hsa-miR-802 31754196 hsa-miR-873-3p 3176 4197 hsa-miR-873-5p 3177 4198 hsa-miR-874 31784199 hsa-miR-875-3p 3179 4200 hsa-miR-875-5p 3180 4201 hsa-miR-876-3p3181 4202 hsa-miR-876-5p 3182 4203 hsa-miR-877-3p 3183 4204hsa-miR-877-5p 3184 4205 hsa-miR-885-3p 3185 4206 hsa-miR-885-5p 31864207 hsa-miR-887 3187 4208 hsa-miR-888-3p 3188 4209 hsa-miR-888-5p 31894210 hsa-miR-889 3190 4211 hsa-miR-890 3191 4212 hsa-miR-891a 3192 4213hsa-miR-891b 3193 4214 hsa-miR-892a 3194 4215 hsa-miR-892b 3195 4216hsa-miR-892c-3p 3196 4217 hsa-miR-892c-5p 3197 4218 hsa-miR-920 31984219 hsa-miR-921 3199 4220 hsa-miR-922 3200 4221 hsa-miR-924 3201 4222hsa-miR-92a-1-5p 3202 4223 hsa-miR-92a-2-5p 3203 4224 hsa-miR-92a-3p3204 4225 hsa-miR-92b-3p 3205 4226 hsa-miR-92b-5p 3206 4227 hsa-miR-9333207 4228 hsa-miR-93-3p 3208 4229 hsa-miR-934 3209 4230 hsa-miR-935 32104231 hsa-miR-93-5p 3211 4232 hsa-miR-936 3212 4233 hsa-miR-937-3p 32134234 hsa-miR-937-5p 3214 4235 hsa-miR-938 3215 4236 hsa-miR-939-3p 32164237 hsa-miR-939-5p 3217 4238 hsa-miR-9-3p 3218 4239 hsa-miR-940 32194240 hsa-miR-941 3220 4241 hsa-miR-942 3221 4242 hsa-miR-943 3222 4243hsa-miR-944 3223 4244 hsa-miR-95 3224 4245 hsa-miR-9-5p 3225 4246hsa-miR-96-3p 3226 4247 hsa-miR-96-5p 3227 4248 hsa-miR-98-3p 3228 4249hsa-miR-98-5p 3229 4250 hsa-miR-99a-3p 3230 4251 hsa-miR-99a-5p 32314252 hsa-miR-99b-3p 3232 4253 hsa-miR-99b-5p 3233 4254

As shown in Table 12, microRNAs are differentially expressed indifferent tissues and cells, and often associated with different typesof diseases (e.g. cancer cells). The decision of removal or insertion ofmicroRNA binding sites, or any combination, is dependent on microRNAexpression patterns and their profilings in cancer cells. In Table 12,“HCC” represents hepatocellular carcinoma, “ALL” stands for acutelymphoblastsic leukemia, “RCC” stands for renal cell carcinoma, “CLL”stands for chrominc lymphocytic leukemia and “MALT” stands formucosa-associated lymphoid tissue.

TABLE 12 mirs, tissues/cell expression and diseases BS mir SEQAssociated Biological microRNA SEQ ID ID Tissues/cells disease functionhsa-let-7a-2-3p 171 1192 Embryonic stem inflammatory, tumor cells, lung,various cancers suppressor, myeloid cells (lung, cervical, breast,pancreatic, etc) hsa-let-7a-3p 172 1193 Embryonic stem inflammatory,tumor cells, lung various cancers suppressor, (lung, cervical, breast,pancreatic, etc) hsa-let-7a-5p 173 1194 Embryonic stem inflammatory,tumor cells, lung various cancers suppressor, (lung, cervical, breast,pancreatic, etc) hsa-let-7b-3p 174 1195 epithelial cells, lung cancer,tumor endothelial cells colorectal cancer, angiogenesis (vascular)cervical cancer, inflammation and immune response after infectionhsa-let-7b-5p 175 1196 epithelial cells, cervical cancer, tumorendothelial cells inflammation and angiogenesis (vascular) immuneresponse after infection hsa-let-7c 176 1197 dendritic cells variouscacners tumor (cervical, suppressor, pancreatic, apoptosis lung,esopphageal, etc) hsa-let-7d-3p 177 1198 embryonic stem associated withtumor cells various cancer suppressor cells hsa-let-7d-5p 178 1199embryonic stem associated with tumor cells various cancer suppressorcells hsa-let-7e-3p 179 1200 immune cells various cancer tumor cells,suppressor autoimmunity, endotoxin tolerance hsa-let-7e-5p 180 1201immune cells various cancer tumor cells suppressor hsa-let-7f-1-3p 1811202 immune cells (T various cancer tumor cells) cells suppressorhsa-let-7f-2-3p 182 1203 immune cells (T various cancer tumor cells)cells suppressor hsa-let-7f-5p 183 1204 immune cells (T Various cancertumor cells) cells suppressor hsa-let-7g-3p 184 1205 hematopoieticvarious cancer tumor cells, adipose, cells (lung, breast, suppressorsmooth muscle etc) cells hsa-let-7g-5p 185 1206 hematopoietic variouscancer tumor cells, adipose, cells (lung, breast, suppressor smoothmuscle etc) cells hsa-let-7i-3p 186 1207 immune cells chronic tumorlymphocyte suppressor leukimia hsa-let-7i-5p 187 1208 immune cellschronic tumor lymphocyte suppressor leukimia hsa-miR-1 188 1209 muscle,heart angiogenesis, cell proliferation(myogenesis) hsa-miR-100-3p 1891210 hematopoietic gastric cancer, tumor cells, endothelial pancreaticcancer angiogenesis cells hsa-miR-100-5p 190 1211 hematopoietic gastriccancer, tumor cells, endothelial pancreatic cancer angiogenesis cellshsa-miR-101-3p 191 1212 endothelial cells various cancers angiogenesis(breast, non-small cell lung, colon, gastric, pancreatic, bladder, etc);lupus erythematosus hsa-miR-101-5p 192 1213 endothelial cells variouscancers angiogenesis (breast, non-small cell lung, colon, gastric,pancreatic, bladder, etc); lupus erythematosus hsa-miR-103a-2-5p 1931214 embryonic stem various cancers oncogene, cell cells, many(endometrial, growth tissues/cells neuroblastoma, colorectal, breast,liver, etc) hsa-miR-103a-3p 194 1215 embryonic stem various cancersoncogene, cell cells, many (endometrial, growth tissues/cellsneuroblastoma, colorectal, breast, liver, etc) hsa-miR-103b 195 1216Many tissues/cells various cancers oncogene, cell (endometrial, growthneuroblastoma, colorectal, breast, liver, etc) hsa-miR-105-3p 196 1217pancreatic cells hsa-miR-105-5p 197 1218 pancreatic cellshsa-miR-106a-3p 198 1219 osteogenic cells osteocarcoma, cell othercancers differentiation hsa-miR-106a-5p 199 1220 osteogenic cellsosteocarcoma, cell other cancers differentiation hsa-miR-106b-3p 2001221 embryonic stem various cancers oncogene cells (non-small lungcancer, gastric cancer, HCC, gliomas, etc) hsa-miR-106b-5p 201 1222embryonic stem various cancers oncogene cells (non-small lung cancer,gastric cancer, HCC, gliomas, etc) hsa-miR-107 202 1223 many tissues,brain breast cancer, hepatocytes/liver pituitary adenoma,obesity/diabetes hsa-miR-10a-3p 203 1224 hematopoeitic acute myeoidoncogene, cell cells leukemia growth hsa-miR-10a-5p 204 1225hematopoeitic acute myeoid oncogene, cell cells leukemia growthhsa-miR-10b-3p 205 1226 multiple tissues various cancers oncogene andcells (breast, ovarian, glioblastoma, pancreatc ductal adenocarcinoma,gastric, etc) hsa-miR-10b-5p 206 1227 multiple tissues various cancersoncogene and cells (breast, ovarian, glioblastoma, pancreatc ductaladenocarcinoma, gastric, etc) hsa-miR-1178-3p 207 1228 osteocarcomahsa-miR-1178-5p 208 1229 osteocarcoma hsa-miR-1179 209 1230 osteocarcomahsa-miR-1180 210 1231 discovered in sarcoma, no expression datahsa-miR-1181 211 1232 downregulated in ovarian cancer cells, associatedwith HCV infection in hepatocytes hsa-miR-1182 212 1233 placentahsa-miR-1183 213 1234 associated with rectal cancer hsa-miR-1184 2141235 Hematopoietic downregulated in cells oral leukoplakia (OLK)hsa-miR-1185-1-3p 215 1236 placenta hsa-miR-1185-2-3p 216 1237 placentahsa-miR-1185-5p 217 1238 placenta hsa-miR-1193 218 1239 melanomahsa-miR-1197 219 1240 neublastoma hsa-miR-1200 220 1241 chroniclynphocytic leukemia hsa-miR-1202 221 1242 chronic lynphocytic leukemia,downregulated in ovarian cancer cells hsa-miR-1203 222 1243 in thechromosome 8q24 region, cancer cells hsa-miR-1204 223 1244 in thechromosome 8q24 region, cancer cells hsa-miR-1205 224 1245 in thechromosome 8q24 region, cancer cells hsa-miR-1206 225 1246 in thechromosome 8q24 region, cancer cells hsa-miR-1207-3p 226 1247 in thechromosome 8q24 region, cancer cells hsa-miR-1207-5p 227 1248 in thechromosome 8q24 region, cancer cells hsa-miR-1208 228 1249 in thechromosome 8q24 region, cancer cells hsa-miR-122-3p 229 1250 kidney,Renal Cell lipid metabolism liver/hepatocytes Carcinoma (RCC), cancercells hsa-miR-1224-3p 230 1251 Lupus nephritis hsa-miR-1224-5p 231 1252rectal cancer hsa-miR-1225-3p 232 1253 adrenal pheochromocytomas;upregulated in MITF KnockDown melanocytes hsa-miR-1225-5p 233 1254prostate cancer hsa-miR-122-5p 234 1255 liver/hepatocytes cancer cellslipid metabolism hsa-miR-1226-3p 235 1256 discovered in a mirtronscreening hsa-miR-1226-5p 236 1257 discovered in a mirtron screeninghsa-miR-1227-3p 237 1258 cartilage/chondrocytes hsa-miR-1227-5p 238 1259cartilage/chondrocytes hsa-miR-1228-3p 239 1260 liver(hepatocytes)Hepatocellular anti-apoptosis carcinoma(HCC) hsa-miR-1228-5p 240 1261liver(hepatocytes) Hepatocellular anti-apoptosis carcinoma(HCC)hsa-miR-1229-3p 241 1262 discovered in a mirtron screeninghsa-miR-1229-5p 242 1263 discovered in a mirtron screening hsa-miR-1231243 1264 HCC hsa-miR-1233-1-5p 244 1265 serum hsa-miR-1233-3p 245 1266serum hsa-miR-1234-3p 246 1267 discovered in embryonic stem cellhsa-miR-1234-5p 247 1268 discovered in embryonic stem cellhsa-miR-1236-3p 248 1269 lymphatic target to endothelial cells VEGFR-3hsa-miR-1236-5p 249 1270 lymphatic target to endothelial cells VEGFR-3hsa-miR-1237-3p 250 1271 esophageal cell line KYSE-150R hsa-miR-1237-5p251 1272 esophageal cell line KYSE-150R hsa-miR-1238-3p 252 1273colorectal cancer hsa-miR-1238-5p 253 1274 colorectal cancerhsa-miR-1243 254 1275 discovered in embryonic stem cells hsa-miR-124-3p255 1276 brain, plasma glioma cell (exosomal) differentiationhsa-miR-1244 256 1277 discovered in embryonic stem cells hsa-miR-1245a257 1278 discovered in embryonic stem cells hsa-miR-1245b-3p 258 1279discovered in embryonic stem cells hsa-miR-1245b-5p 259 1280 discoveredin embryonic stem cells hsa-miR-124-5p 260 1281 brain, Plasmaupregulated in cell (circulating) heart dysfunction, differentiationglioma hsa-miR-1246 261 1282 embryonic stem cells, epithelial cellshsa-miR-1247-3p 262 1283 embryoid body cells hsa-miR-1247-5p 263 1284embryoid body cells hsa-miR-1248 264 1285 component of SnoRNAshsa-miR-1249 265 1286 liver(hepatocytes) hsa-miR-1250 266 1287oligodendrocytes hsa-miR-1251 267 1288 discovered in embryonic stemcells hsa-miR-1252 268 1289 discovered in embryonic stem cellshsa-miR-1253 269 1290 discovered in embryonic stem cells hsa-miR-1254270 1291 embryonic stem cells hsa-miR-1255a 271 1292 discovered inembryonic stem cells hsa-miR-1255b-2- 272 1293 discovered in 3pembryonic stem cells hsa-miR-1255b-5p 273 1294 discovered in embryonicstem cells hsa-miR-1256 274 1295 discovered in prostate cancer embryonicstem cells hsa-miR-1257 275 1296 discovered in liposarcoma (softembryonic stem tissue sarcoma) cells hsa-miR-1258 276 1297 discovered inbreast cancer and embryonic stem lung cancer cells hsa-miR-125a-3p 2771298 brain, various cancer cell proliferation hematopoietic (prostate,HCC, and cells etc) differentiation hsa-miR-125a-5p 278 1299 brain,various cancer cell proliferation hematopoietic (prostate, HCC, andcells etc) differentiation hsa-miR-125b-1-3p 279 1300 hematopoieticvarious cancer oncogene, cell cells (monocytes), (prostate, HCC,differentiation brain(neuron) etc) hsa-miR-125b-2-3p 280 1301hematopoietic various cancer oncogene, cell cells (monocytes),(prostate, HCC, differentiation brain(neuron) etc) hsa-miR-125b-5p 2811302 hematopoietic various cancer oncogene, cell cells, brain (cutaneousT cell differentiation (neuron) lymphoma, prostate, HCC, etc)hsa-miR-1260a 282 1303 periodontal tissue hsa-miR-1260b 283 1304periodontal tissue hsa-miR-1261 284 1305 embryonic stem cellshsa-miR-1262 285 1306 embryoid body cells hsa-miR-1263 286 1307discovered in embryonic stem cells hsa-miR-126-3p 287 1308 endothelialB-lieage ALL angiogenesis cells, lung hsa-miR-1264 288 1309 discoveredin embryonic stem cells hsa-miR-1265 289 1310 discovered in embryonicstem cells hsa-miR-126-5p 290 1311 endothelial breast cancer, B-angiogenesis cells, lung lieage ALL hsa-miR-1266 291 1312 embryonic stemcells hsa-miR-1267 292 1313 discovered in embryonic stem cellshsa-miR-1268a 293 1314 embryonic stem cells hsa-miR-1268b 294 1315embryonic stem cells hsa-miR-1269a 295 1316 embryoid body cellshsa-miR-1269b 296 1317 embryoid body cells hsa-miR-1270 297 1318discovered in embryonic stem cells hsa-miR-1271-3p 298 1319 brainHepatocellular Suppress GPC-3 carcinoma(HCC) in HCC hsa-miR-1271-5p 2991320 brain Hepatocellular Suppress GPC-3 carcinoma(HCC) in HCChsa-miR-1272 300 1321 embryonic stem cells hsa-miR-1273a 301 1322discovered in embryonic stem cells hsa-miR-1273c 302 1323 colorectalcancer hsa-miR-1273d 303 1324 discovered in embryonic stem cellshsa-miR-1273e 304 1325 solid tumor cells hsa-miR-1273f 305 1326 cervicalcancer hsa-miR-1273g-3p 306 1327 cervical cancer hsa-miR-1273g-5p 3071328 cervical cancer hsa-miR-127-3p 308 1329 lung, placenta hsa-miR-1275309 1330 embryonic stem gastric carcinoma cells hsa-miR-127-5p 310 1331lung, placenta(islet) hsa-miR-1276 311 1332 discovered in embryonic stemcells hsa-miR-1277-3p 312 1333 embryoid body cells hsa-miR-1277-5p 3131334 embryoid body cells hsa-miR-1278 314 1335 discovered in embryonicstem cells hsa-miR-1279 315 1336 monocytes hsa-miR-128 316 1337glioblast, brain B-lieage ALL target to neurofibrominlin neuronhsa-miR-1281 317 1338 muscle invasive bladder cancer hsa-miR-1282 3181339 discovered in embryonic stem cells hsa-miR-1283 319 1340 placentahsa-miR-1284 320 1341 lung cancer hsa-miR-1285-3p 321 1342 variouscancer inhibit P53 cells expression hsa-miR-1285-5p 322 1343 variouscancer inhibit P53 cells expression hsa-miR-1286 323 1344 smooth muscleesophageal cancer hsa-miR-1287 324 1345 embryoid body breast cancercells hsa-miR-1288 325 1346 discovered in embryonic stem cellshsa-miR-1289 326 1347 multiple cell types hsa-miR-1290 327 1348 embryoidbody gastric carcinoma cells hsa-miR-1291 328 1349 hepatocytes componentof SnoRNAs hsa-miR-129-1-3p 329 1350 multiple cell types HCC cancercells hsa-miR-1292-3p 330 1351 hsa-miR-129-2-3p 331 1352 multiple celltypes various cancer cells hsa-miR-1292-5p 332 1353 hsa-miR-1293 3331354 discovered in embryonic stem cells hsa-miR-1294 334 1355 discoveredin embryonic stem cells hsa-miR-1295a 335 1356 tumor cells (follicularlymphoma) hsa-miR-1295b-3p 336 1357 tumor cells (follicular lymphoma)hsa-miR-1295b-5p 337 1358 tumor cells (follicular lymphoma)hsa-miR-129-5p 338 1359 liver(hepatocytes) HCC, thyroid cell death incancer cancer cell hsa-miR-1296 339 1360 breast cancer hsa-miR-1297 3401361 discovered in embryonic stem cells hsa-miR-1298 341 1362hsa-miR-1299 342 1363 discovered in embryonic stem cells hsa-miR-1301343 1364 breast cancer hsa-miR-1302 344 1365 hsa-miR-1303 345 1366hepatocyte colorectal cancer, liver cancer hsa-miR-1304-3p 346 1367dental development hsa-miR-1304-5p 347 1368 dental developmenthsa-miR-1305 348 1369 discovered in embryonic stem cells hsa-miR-1306-3p349 1370 discovered in embryonic stem cells hsa-miR-1306-5p 350 1371discovered in embryonic stem cells hsa-miR-1307-3p 351 1372 discoveredin embryonic stem cells hsa-miR-1307-5p 352 1373 discovered in embryonicstem cells hsa-miR-130a-3p 353 1374 lung, monocytes, various cancerspro-angiogenic vascular (basal cell endothelial cells carcinoma, HCC,ovarian, etc), drug resistance hsa-miR-130a-5p 354 1375 lung, monocytes,various cancers pro-angiogenic vascular (basal cell endothelial cellscarcinoma, HCC, ovarian, etc), drug resistance hsa-miR-130b-3p 355 1376Lung, epidermal various cancers cell cells (gastric, rena cellproiferation/senescence (keratinocytes) carcinoma) hsa-miR-130b-5p 3561377 Lung, epidermal various cancers cell cells (gastric, rena cellproiferation/senescence (keratinocytes) carcinoma) hsa-miR-1321 357 1378neuroblastoma hsa-miR-1322 358 1379 neuroblastoma hsa-miR-1323 359 1380placenta neuroblastoma hsa-miR-132-3p 360 1381 Brain(neuron), immunecells hsa-miR-1324 361 1382 neuroblastoma hsa-miR-132-5p 362 1383brain(neuron), immune cells hsa-miR-133a 363 1384 muscle, heart, heartfailure, myogenesis epithelial cells esophageal cancer (lung)hsa-miR-133b 364 1385 muscle, heart, heart failure, myogenesisepithelial cells esophageal cancer (lung) hsa-miR-134 365 1386 lung(epithelial) non-samll cell lung cancer, pulmonary embolism hsa-miR-1343366 1387 breast cancer cells hsa-miR-135a-3p 367 1388 brain, othertissues various cancer tumor cells (lung, breast, suppressor colorectal,HCC, etc) hsa-miR-135a-5p 368 1389 brain, other tissues various cancertumor cells (lung, breast, suppressor colorectal, HCC, etc)hsa-miR-135b-3p 369 1390 brain, placenta, various cancers other tissues(gastric, mammary, neuroblastomas, pancreatic, etc) hsa-miR-135b-5p 3701391 brain, placenta, various cancers other tissues (gastric, mammary,neuroblastomas, pancreatic, etc) hsa-miR-136-3p 371 1392 stem cells,glioma tumor placenta suppressor hsa-miR-136-5p 372 1393 stem cells,glioma tumor placenta suppressor hsa-miR-137 373 1394 brain variouscancers inhibiting cancer (glioblastoma, cell proliferation breast,gastric etc), and migration Alzheimer's disease hsa-miR-138-1-3p 3741395 stem cells, arious cancer cells, cell epidermal downregulated inproliferation/senescence cells(keratinocytes) HCC hsa-miR-138-2-3p 3751396 stem cells arious cancer cells, downregulated in HCC hsa-miR-138-5p376 1397 stem cells arious cancer cells, downregulated in HCChsa-miR-139-3p 377 1398 hematocytes, brain various cancer repress cancercells (colorectal, metastasis gastric, ovarian) hsa-miR-139-5p 378 1399hematocytes, brain various cancer repress cancer cells (colorectal,metastasis gastric, ovarian) hsa-miR-140-3p 379 1400 airway smooth Virusinfection, muscle cancers hsa-miR-140-5p 380 1401 cartilage csncers(chondrocytes) hsa-miR-141-3p 381 1402 Many tissues/cells various cancercell cells (HCC, differentiation prostate, kidney, etc) hsa-miR-141-5p382 1403 Many tissues/cells various cancer cell cells (HCC,differentiation prostate, kidney, etc) hsa-miR-142-3p 383 1404 meyloidcells, immune hematopoiesis, response APC cells hsa-miR-142-5p 384 1405meyloid cells, immune hematopoiesis, response APC cells hsa-miR-143-3p385 1406 vascular smooth pre-B-cell acute muscle lymphocytic leukemia,virus infection hsa-miR-143-5p 386 1407 vascular smooth virus infectionmuscle, T-cells hsa-miR-144-3p 387 1408 erythroid various cancers cell(lung, colorectal, differentiation etc) hsa-miR-144-5p 388 1409erythroid various cancers cell (lung, colorectal, differentiation etc)hsa-miR-145-3p 389 1410 kidney, cartilage, T-cell lupus tumor vascularsmooth suppressor muscle hsa-miR-145-5p 390 1411 kidney, cartilage,T-cell lupus tumor vascular smooth suppressor muscle hsa-miR-1468 3911412 lung cancer hsa-miR-1469 392 1413 tumor cell(follicular lymphoma),rectal cancer hsa-miR-146a-3p 393 1414 immune cells, various cancers,hematopoiesis endotoxin tolerance hsa-miR-146a-5p 394 1415 immune cells,various cancers, hematopoiesis endotoxin tolerance hsa-miR-146b-3p 3951416 immune cells various cancers hsa-miR-146b-5p 396 1417 Embryonicstem various cancers tumor invation, cells (glioma) migrationhsa-miR-1470 397 1418 hsa-miR-1471 398 1419 tumor cell(follicularlymphoma), rectal cancer hsa-miR-147a 399 1420 Macrophage inflammatoryresponse hsa-miR-147b 400 1421 Macrophage inflammatory responsehsa-miR-148a-3p 401 1422 hematopoietic CLL, T-lineage cells ALLhsa-miR-148a-5p 402 1423 hematopoietic CLL, T-lineage cells ALLhsa-miR-148b-3p 403 1424 neuron hsa-miR-148b-5p 404 1425 neuronhsa-miR-149-3p 405 1426 heart, brain various cancers (glioma,colorectal, gastric, etc) hsa-miR-149-5p 406 1427 heart, brain variouscancers (glioma, colorectal, gastric, etc) hsa-miR-150-3p 407 1428hematopoietic circulating plasma cells (lymphoid) (acute myeloidleukemia) hsa-miR-150-5p 408 1429 hematopoietic circulating plasma cells(lymphoid) (acute myeloid leukemia) hsa-miR-151a-3p 409 1430 neuron,fetal liver hsa-miR-151a-5p 410 1431 neuron, fetal liver hsa-miR-151b411 1432 immune cells (B- cells) hsa-miR-152 412 1433 liver hsa-miR-153413 1434 brain hsa-miR-1537 414 1435 hsa-miR-1538 415 1436 blood Cancercells hsa-miR-1539 416 1437 esophageal cell line KYSE-150Rhsa-miR-154-3p 417 1438 embryonic stem cells hsa-miR-154-5p 418 1439embryonic stem cells hsa-miR-155-3p 419 1440 T/B cells, various cancersmonocytes, breast (CLL, B cell lymphoma, breast, lung, ovarian,cervical, colorectal, prostate) hsa-miR-155-5p 420 1441 T/B cells,various cancers monocytes, breast (CLL, B cell lymphoma, breast, lung,ovarian, cervical, colorectal, prostate) hsa-miR-1587 421 1442identified in B- cells hsa-miR-15a-3p 422 1443 blood, cell cycle,lymphocyte, proliferation hematopoietic tissues (spleen) hsa-miR-15a-5p423 1444 blood, cell cycle, lymphocyte, proliferation hematopoietictissues (spleen) hsa-miR-15b-3p 424 1445 blood, cell cycle, lymphocyte,proliferation hematopoietic tissues (spleen) hsa-miR-15b-5p 425 1446blood, cell cycle, lymphocyte, proliferation hematopoietic tissues(spleen) hsa-miR-16-1-3p 426 1447 embryonic stem cells, blood,hematopoietic tissues (spleen) hsa-miR-16-2-3p 427 1448 blood,lymphocyte, hematopoietic tissues (spleen) hsa-miR-16-5p 428 1449 Manytissues, blood hsa-miR-17-3p 429 1450 embryonic stem tumor cells,endothelial angiogenesis cells, hsa-miR-17-5p 430 1451 endothelialcells, tumor kidney, breast; angiogenesis hsa-miR-181a-2-3p 431 1452glioblast, stem cells hsa-miR-181a-3p 432 1453 glioblast, myeloid cells,Embryonic stem cells hsa-miR-181a-5p 433 1454 glioblast, myeloid cells,Embryonic stem cells hsa-miR-181b-3p 434 1455 glioblast, cell Embryonicstem proiferation/senescence cells, epidermal (keratinocytes)hsa-miR-181b-5p 435 1456 glioblast, cell Embryonic stemproiferation/senescence cells, epidermal (keratinocytes) hsa-miR-181c-3p436 1457 brain, stem variou cance cells cell cells/progenitor(gliobasltoma, differentiation basal cell carcinoma, prostate)hsa-miR-181c-5p 437 1458 brain, stem variou cance cells cellcells/progenitor (gliobasltoma, differentiation basal cell carcinoma,prostate) hsa-miR-181d 438 1459 glia cells hsa-miR-182-3p 439 1460immune cells autoimmune immune response hsa-miR-1825 440 1461 discoveredin a MiRDeep screening hsa-miR-182-5p 441 1462 lung, immune cellsautoimmune immune response hsa-miR-1827 442 1463 small cell lung cancerhsa-miR-183-3p 443 1464 brain hsa-miR-183-5p 444 1465 brain hsa-miR-184445 1466 blood, tongue, pancreas (islet) hsa-miR-185-3p 446 1467hsa-miR-185-5p 447 1468 hsa-miR-186-3p 448 1469 osteoblasts, heartvarious cancer cells hsa-miR-186-5p 449 1470 osteoblasts, heart variouscancer cells hsa-miR-187-3p 450 1471 thyroid tumor hsa-miR-187-5p 4511472 thyroid tumor hsa-miR-188-3p 452 1473 irway smooth muscle, centralnervous system hsa-miR-188-5p 453 1474 irway smooth muscle, centralnervous system hsa-miR-18a-3p 454 1475 endothelial cells, lunghsa-miR-18a-5p 455 1476 endothelial cells, lung hsa-miR-18b-3p 456 1477lung hsa-miR-18b-5p 457 1478 lung hsa-miR-1908 458 1479 breast cancerhsa-miR-1909-3p 459 1480 rectal cancer hsa-miR-1909-5p 460 1481 rectalcancer hsa-miR-190a 461 1482 brain hsa-miR-190b 462 1483 brainhsa-miR-1910 463 1484 embryonic stem cells hsa-miR-1911-3p 464 1485embryonic stem cells, neural precursor hsa-miR-1911-5p 465 1486embryonic stem cells, neural precursor hsa-miR-1912 466 1487 embryonicstem cells, neural precursor hsa-miR-1913 467 1488 embryonic stem cellshsa-miR-191-3p 468 1489 chroninc lymphocyte leukimia, B-lieage ALLhsa-miR-1914-3p 469 1490 embryonic stem cells hsa-miR-1914-5p 470 1491embryonic stem cells hsa-miR-1915-3p 471 1492 embryonic stem cellshsa-miR-1915-5p 472 1493 embryonic stem cells hsa-miR-191-5p 473 1494chroninc lymphocyte leukimia, B-lieage ALL hsa-miR-192-3p 474 1495kidney hsa-miR-192-5p 475 1496 kidney hsa-miR-193a-3p 476 1497 manytissues/cells various cancer tumor cells (lung, suppressor,osteoblastoma, proliferation ALL, follicular lymphoma, etc)hsa-miR-193a-5p 477 1498 many tissues/cells various cancer tumor cells(lung, suppressor, osteoblastoma, proliferation ALL, follicularlymphoma, etc) hsa-miR-193b-3p 478 1499 many tissues/cells, ariouscancer cells tumor semen (prostate, breast, suppressor melanoma,myeloma, non small cell lung, etc)follicular lymphoma) hsa-miR-193b-5p479 1500 many tissues/cells, arious cancer cells tumor semen (prostate,breast, suppressor melanoma, myeloma, non small cell lung,etc)follicular lymphoma) hsa-miR-194-3p 480 1501 kidney, liver variouscancers hsa-miR-194-5p 481 1502 kidney, liver various cancershsa-miR-195-3p 482 1503 breast, pancreas (islet) hsa-miR-195-5p 483 1504breast, pancreas (islet) hsa-miR-196a-3p 484 1505 pancreatic variouscancer oncogenic, cells, endometrial cells (pancreatic, tumor tissues,osteosarcoma, suppressor mesenchymal endometrial, AML stem cells etc)hsa-miR-196a-5p 485 1506 pancreatic various cancer oncogenic, cells,endometrial cells (pancreatic, tumor tissues, osteosarcoma, suppressormesenchymal endometrial, AML stem cells etc) hsa-miR-196b-3p 486 1507endometrial tissues glioblastoma apoptosis hsa-miR-196b-5p 487 1508endometrial tissues glioblastoma apoptosis hsa-miR-1972 488 1509 acutelymphoblastic leukemia hsa-miR-1973 489 1510 acute lymphoblasticleukemia hsa-miR-197-3p 490 1511 blood (myeloid), various cancers othertissues/cells (thyroid tumor, leukemia, etc) hsa-miR-197-5p 491 1512blood (myeloid), various cancers other tissues/cells (thyroid tumor,leukemia, etc) hsa-miR-1976 492 1513 acute lymphoblastic leukemiahsa-miR-198 493 1514 central nevous system(CNS) hsa-miR-199a-3p 494 1515liver, embryoid body cells, cardiomyocytes hsa-miR-199a-5p 495 1516liver, cardiomyocytes hsa-miR-199b-3p 496 1517 liver, osteoblast variouscancers osteogenesis hsa-miR-199b-5p 497 1518 liver, osteoblast variouscancers osteogenesis hsa-miR-19a-3p 498 1519 endothelial cells tumorangiogenesis hsa-miR-19a-5p 499 1520 endothelial cells tumorangiogenesis hsa-miR-19b-1-5p 500 1521 endothelial cells tumorangiogenesis hsa-miR-19b-2-5p 501 1522 endothelial cells tumorangiogenesis hsa-miR-19b-3p 502 1523 endothelial cells tumorangiogenesis hsa-miR-200a-3p 503 1524 epithelial cells, various cancerstumor many other tissues (breast, cervical, progression and bladder,etc) metastasis hsa-miR-200a-5p 504 1525 epithelial cells, variouscancers tumor many other tissues (breast, cervical, progression andbladder, etc) metastasis hsa-miR-200b-3p 505 1526 epithelial cells,tumor many other tissues progression and metastasis hsa-miR-200b-5p 5061527 epithelial cells, tumor many other tissues progression andmetastasis hsa-miR-200c-3p 507 1528 epithelial cells, tumor many othertissues, progression and embryonic stem metastasis cells hsa-miR-200c-5p508 1529 epithelial cells, tumor many other tissues, progression andembryonic stem metastasis cells hsa-miR-202-3p 509 1530 bloodlymphomagenesis, other cancers hsa-miR-202-5p 510 1531 bloodlymphomagenesis, other cancers hsa-miR-203a 511 1532 skin (epithelium)psoriasis, autoimmune hsa-miR-203b-3p 512 1533 skin specific psoriasis,(epithelium) autoimmune hsa-miR-203b-5p 513 1534 skin specificpsoriasis, (epithelium) autoimmune hsa-miR-204-3p 514 1535 adipose,other various cancers tumor metastasis tissues/cells. kidneyhsa-miR-204-5p 515 1536 adipose, other various cancers tumor metastasistissues/cells, kidney hsa-miR-2052 516 1537 hsa-miR-2053 517 1538hsa-miR-205-3p 518 1539 blood(plasma) various cancer cells (breast,glioma, melanoma, endometrial, etc) hsa-miR-2054 519 1540 hsa-miR-205-5p520 1541 blood(plasma) various cancer cells (breast, glioma, melanoma,endometrial, etc) hsa-miR-206 521 1542 muscle (cardiac myogenesis andskeletal) hsa-miR-208a 522 1543 heart(cardiomyocyte), cardiac defectsmuscle hsa-miR-208b 523 1544 heart(cardiomyocyte), cardiac defectsmuscle hsa-miR-20a-3p 524 1545 endothelial cells, kidney, osteogeniccells hsa-miR-20a-5p 525 1546 endothelial cells, kidney, osteogeniccells hsa-miR-20b-3p 526 1547 osteogenic cells hsa-miR-20b-5p 527 1548osteogenic cells hsa-miR-210 528 1549 kidney, heart, RCC, B-cellangiogenesis vascular lymphocytes endothelial cells hsa-miR-2110 5291550 rectal cancer hsa-miR-2113 530 1551 embryonic stem cellshsa-miR-211-3p 531 1552 melanocytes melanoma and other cancershsa-miR-2114-3p 532 1553 ovary, female reproductuve tracthsa-miR-2114-5p 533 1554 ovary, female reproductuve tracthsa-miR-2115-3p 534 1555 female ovarian cancer reproductive tracthsa-miR-2115-5p 535 1556 female ovarian cancer reproductive tracthsa-miR-211-5p 536 1557 melanocytes melanoma and other cancershsa-miR-2116-3p 537 1558 live cancer(hepatocytes) and ovarian cancerhsa-miR-2116-5p 538 1559 live cancer(hepatocytes) and ovarian cancerhsa-miR-2117 539 1560 ovarian cancer hsa-miR-212-3p 540 1561brain(neuron), lymphoma spleen hsa-miR-212-5p 541 1562 brain(neuron),lymphoma spleen hsa-miR-21-3p 542 1563 glioblast, Blood autoimmune,heart (meyloid cells), diseases, cancers liver, vascular endothelialcells hsa-miR-214-3p 543 1564 immune cerlls, varioua cancers immunepancreas (melanoma, response pancreatic, ovarian) hsa-miR-214-5p 5441565 immune cells, varioua cancers immune pancreas (melanoma, responsepancreatic, ovarian) hsa-miR-215 545 1566 many tissues/cells variouscancers cell cycle (renal, colon, arrest/p53 osteosarcoma) induciblehsa-miR-21-5p 546 1567 blood (myeloid autoimmune, heart cells), liver,diseases, cancers endothelial cells hsa-miR-216a-3p 547 1568 kidney,pancreas hsa-miR-216a-5p 548 1569 kidney, pancreas hsa-miR-216b 549 1570cancers senescence hsa-miR-217 550 1571 endothelial cells various cancercells (pancreas, kidney, breast) hsa-miR-218-1-3p 551 1572 endothelialcells various cancer cells (gastric tumor, bladder, cervical, etc)hsa-miR-218-2-3p 552 1573 various cancer cells (gastric tumor, bladder,cervical, etc) hsa-miR-218-5p 553 1574 various cancer cells (gastrictumor, bladder, cervical, etc) hsa-miR-219-1-3p 554 1575 brain,oligodendrocytes hsa-miR-219-2-3p 555 1576 brain, oligodendrocyteshsa-miR-219-5p 556 1577 brain, oligodendrocytes hsa-miR-221-3p 557 1578endothelial cells, leukemia and other angiogenesis/vasculogenesis immunecells cancers hsa-miR-221-5p 558 1579 endothelial cells, leukemia andother angiogenesis/vasculogenesis immune cells cancers hsa-miR-222-3p559 1580 endothelial cells various cancers angiogenesis hsa-miR-222-5p560 1581 endothelial cells various cancers angiogenesis hsa-miR-223-3p561 1582 meyloid cells leukemia hsa-miR-223-5p 562 1583 meyloid cellsleukemia hsa-miR-22-3p 563 1584 many tissues/cells various cancerstumorigenesis hsa-miR-224-3p 564 1585 blood(plasma), cancers and ovaryinflammation hsa-miR-224-5p 565 1586 blood(plasma), cancers and ovaryinflammation hsa-miR-22-5p 566 1587 many tissues/cells Various cancerstumorigenesis hsa-miR-2276 567 1588 breast cancer hsa-miR-2277-3p 5681589 female reproductive tract hsa-miR-2277-5p 569 1590 femalereproductive tract hsa-miR-2278 570 1591 breast cancer hsa-miR-2355-3p571 1592 embryonic stem cells hsa-miR-2355-5p 572 1593 embryonic stemcells hsa-miR-2392 573 1594 identified in B- cells hsa-miR-23a-3p 5741595 brain(astrocyte), Cancers endothelial cells, blood(erythroid)hsa-miR-23a-5p 575 1596 brain(astrocyte), cancers endothelial cells,blood(erythroid) hsa-miR-23b-3p 576 1597 blood, meyloid cancers (renalcells cancer, glioblastoma, prostate, etc) and autoimmune hsa-miR-23b-5p577 1598 blood, meyloid cancers(glioblastoma, cells prostate, etc) andautoimmune hsa-miR-23c 578 1599 cervical cancer hsa-miR-24-1-5p 579 1600lung, meyloid cells hsa-miR-24-2-5p 580 1601 lung, meyloid cellshsa-miR-24-3p 581 1602 lung, meyloid cells hsa-miR-2467-3p 582 1603breast cancer hsa-miR-2467-5p 583 1604 breast cancer hsa-miR-25-3p 5841605 embryonic stem cells, airway smooth muscle hsa-miR-25-5p 585 1606embryonic stem cells, airway smooth muscle hsa-miR-2681-3p 586 1607breast cancer hsa-miR-2681-5p 587 1608 breast cancer hsa-miR-2682-3p 5881609 hsa-miR-2682-5p 589 1610 hsa-miR-26a-1-3p 590 1611 embryonic stemCLL and other cell cycle and cells, blood, other cancers differentiationtissues hsa-miR-26a-2-3p 591 1612 blood, other CLL and other cell cycleand tissues cancers differentiation hsa-miR-26a-5p 592 1613 blood, otherCLL and other cell cycle and tissues cancers differentiationhsa-miR-26b-3p 593 1614 hematopoietic cells hsa-miR-26b-5p 594 1615hematopoietic cells hsa-miR-27a-3p 595 1616 meyloid cells various cancercells hsa-miR-27a-5p 596 1617 meyloid cells various cancer cellshsa-miR-27b-3p 597 1618 meyloid cells, various cancer pro-angiogenicvascular cells endothelial cells hsa-miR-27b-5p 598 1619 meyloid cells,various cancer pro-angiogenic vascular cells endothelial cellshsa-miR-28-3p 599 1620 blood(immune B/T cell cells) lymphomahsa-miR-28-5p 600 1621 blood(immune B/T cell cells) lymphomahsa-miR-2861 601 1622 osteoblasts basal cell carcinoma hsa-miR-2909 6021623 T-Lymphocytes hsa-miR-296-3p 603 1624 kidney, heart, lung,angiogenesis entothelial cells hsa-miR-2964a-3p 604 1625hsa-miR-2964a-5p 605 1626 hsa-miR-296-5p 606 1627 lung, liver,angiogenesis endothelial cells hsa-miR-297 607 1628 oocyte and prostatehsa-miR-298 608 1629 breast cancer hsa-miR-299-3p 609 1630 myeloidleukaemia, hepatoma, breast cancer hsa-miR-299-5p 610 1631 myeloidleukaemia, hepatoma, breast cancer hsa-miR-29a-3p 611 1632 immuno systemCLL, other tumor cancers, suppression, neurodegenative immune diseasemodulation hsa-miR-29a-5p 612 1633 immuno system CLL, other tumorcancers, suppression, neurodegenative immune disease modulationhsa-miR-29b-1-5p 613 1634 immuno system CLL, other tumor cancers,suppression, neurodegenative immune disease modulation hsa-miR-29b-2-5p614 1635 immuno system CLL, other cancers tumor suppression, immunemodulation hsa-miR-29b-3p 615 1636 immuno system CLL, other cancerstumor suppression, immune modulation hsa-miR-29c-3p 616 1637 immunosystem CLL, other cancers tumor suppression, immune modulationhsa-miR-29c-5p 617 1638 immuno system CLL, other cancers tumorsuppression, immune modulation hsa-miR-300 618 1639 osteoblast Bladdercancer hsa-miR-301a-3p 619 1640 embryonic stem cells hsa-miR-301a-5p 6201641 embryonic stem cells hsa-miR-301b 621 1642 esophagealadenocarcinoma, colonic cancer hsa-miR-302a-3p 622 1643 embryonic stemlipid metabolism cells, lipid metabolism hsa-miR-302a-5p 623 1644embryonic stem lipid metabolism cells, lipid metabolism hsa-miR-302b-3p624 1645 embryonic stem cells hsa-miR-302b-5p 625 1646 embryonic stemcells hsa-miR-302c-3p 626 1647 embryonic stem cells hsa-miR-302c-5p 6271648 embryonic stem cells hsa-miR-302d-3p 628 1649 embryonic stem cellshsa-miR-302d-5p 629 1650 embryonic stem cells hsa-miR-302e 630 1651embryoid body cells hsa-miR-302f 631 1652 gastric cancer hsa-miR-3064-3p632 1653 hsa-miR-3064-5p 633 1654 hsa-miR-3065-3p 634 1655oligodendrocytes anti-virus response hsa-miR-3065-5p 635 1656oligodendrocytes solid tumors hsa-miR-3074-3p 636 1657 variouscancer(melanoma, breast) hsa-miR-3074-5p 637 1658 variouscancer(melanoma, breast) hsa-miR-30a-3p 638 1659 kidney, pancreaticvarious cancers autophagy cells hsa-miR-30a-5p 639 1660 CNS(prefrontalglioma, colon autophagy cortex), other carcinoma tissues hsa-miR-30b-3p640 1661 kidney, adipose, CNS(prefrontal cortex) hsa-miR-30b-5p 641 1662kidney, adipose, CNS(prefrontal cortex) hsa-miR-30c-1-3p 642 1663kidney, adipose, CNS(prefrontal cortex) hsa-miR-30c-2-3p 643 1664kidney, adipose, CNS(prefrontal cortex) hsa-miR-30c-5p 644 1665 kidney,adipose, CNS(prefrontal cortex) hsa-miR-30d-3p 645 1666 CNS (prefrontalcortex hsa-miR-30d-5p 646 1667 CNS (prefrontal cortex, embryoid bodycells hsa-miR-30e-3p 647 1668 myeloid cells, glia cells hsa-miR-30e-5p648 1669 myeloid cells, glia cells hsa-miR-3115 649 1670 various cancer(melanoma, breast tumor) hsa-miR-3116 650 1671 discovered in themelanoma miRNAome hsa-miR-3117-3p 651 1672 discovered in the melanomamiRNAome hsa-miR-3117-5p 652 1673 discovered in the melanoma miRNAomehsa-miR-3118 653 1674 discovered in the melanoma miRNAome hsa-miR-3119654 1675 discovered in the melanoma miRNAome hsa-miR-3120-3p 655 1676discovered in the breast tumor melanoma miRNAome hsa-miR-3120-5p 6561677 discovered in the breast tumor melanoma miRNAome hsa-miR-3121-3p657 1678 discovered in the breast tumor melanoma miRNAomehsa-miR-3121-5p 658 1679 discovered in the breast tumor melanomamiRNAome hsa-miR-3122 659 1680 discovered in the melanoma miRNAomehsa-miR-3123 660 1681 discovered in the melanoma miRNAomehsa-miR-3124-3p 661 1682 discovered in the breast tumor melanomamiRNAome, ovary hsa-miR-3124-5p 662 1683 discovered in the breast tumormelanoma miRNAome, ovary hsa-miR-3125 663 1684 discovered in themelanoma miRNAome hsa-miR-3126-3p 664 1685 discovered in the breasttumor melanoma miRNAome, ovary hsa-miR-3126-5p 665 1686 discovered inthe breast tumor melanoma miRNAome, ovary hsa-miR-3127-3p 666 1687discovered in the breast tumor melanoma miRNAome hsa-miR-3127-5p 6671688 discovered in the breast tumor melanoma miRNAome hsa-miR-3128 6681689 discovered in the breast tumor melanoma miRNAome hsa-miR-3129-3p669 1690 discovered in the breast tumor melanoma miRNAome, ovaryhsa-miR-3129-5p 670 1691 discovered in the breast tumor melanomamiRNAome, ovary hsa-miR-3130-3p 671 1692 discovered in the breast tumormelanoma miRNAome, ovary hsa-miR-3130-5p 672 1693 discovered in thebreast tumor melanoma miRNAome, ovary hsa-miR-3131 673 1694 discoveredin the breast tumor melanoma miRNAome hsa-miR-3132 674 1695 discoveredin the melanoma miRNAome hsa-miR-3133 675 1696 discovered in themelanoma miRNAome hsa-miR-3134 676 1697 discovered in the melanomamiRNAome hsa-miR-3135a 677 1698 discovered in the melanoma miRNAomehsa-miR-3135b 678 1699 discovered in B cells hsa-miR-3136-3p 679 1700discovered in the lymphoblastic melanoma leukaemia and miRNAome breasttumor hsa-miR-3136-5p 680 1701 discovered in the lymphoblastic melanomaleukaemia and miRNAome breast tumor hsa-miR-3137 681 1702 discovered inthe melanoma miRNAome hsa-miR-3138 682 1703 discovered in the melanomamiRNAome, ovary hsa-miR-3139 683 1704 discovered in the melanomamiRNAome hsa-miR-31-3p 684 1705 hsa-miR-3140-3p 685 1706 discovered inthe lymphoblastic melanoma leukaemia and miRNAome, ovary breast tumorhsa-miR-3140-5p 686 1707 discovered in the lymphoblastic melanomaleukaemia and miRNAome, ovary breast tumor hsa-miR-3141 687 1708discovered in the melanoma miRNAome hsa-miR-3142 688 1709 discovered inthe melanoma miRNAome; immune cells hsa-miR-3143 689 1710 discovered inthe breast tumor melanoma miRNAome hsa-miR-3144-3p 690 1711 discoveredin the melanoma miRNAome, ovary hsa-miR-3144-5p 691 1712 discovered inthe melanoma miRNAome, ovary hsa-miR-3145-3p 692 1713 discovered in thebreast tumor melanoma miRNAome hsa-miR-3145-5p 693 1714 discovered inthe breast tumor melanoma miRNAome hsa-miR-3146 694 1715 discovered inthe breast tumor melanoma miRNAome hsa-miR-3147 695 1716 discovered inthe melanoma miRNAome hsa-miR-3148 696 1717 discovered in the melanomamiRNAome hsa-miR-3149 697 1718 discovered in the melanoma miRNAome,ovary hsa-miR-3150a-3p 698 1719 discovered in the breast tumor melanomamiRNAome hsa-miR-3150a-5p 699 1720 discovered in the breast tumormelanoma miRNAome hsa-miR-3150b-3p 700 1721 discovered in the breasttumor and melanoma lymphoblastic miRNAome leukaemia hsa-miR-3150b-5p 7011722 discovered in the breast tumor and melanoma lymphoblastic miRNAomeleukaemia hsa-miR-3151 702 1723 discovered in the lymphoblastic melanomaleukaemia miRNAome hsa-miR-3152-3p 703 1724 discovered in the breasttumor melanoma miRNAome, ovary hsa-miR-3152-5p 704 1725 discovered inthe breast tumor melanoma miRNAome, ovary hsa-miR-3153 705 1726discovered in the melanoma miRNAome hsa-miR-3154 706 1727 discovered inthe lymphoblastic melanoma leukaemia miRNAome hsa-miR-3155a 707 1728discovered in the melanoma miRNAome hsa-miR-3155b 708 1729 discovered inB cells hsa-miR-3156-3p 709 1730 discovered in the breast tumor melanomamiRNAome hsa-miR-3156-5p 710 1731 discovered in the breast tumormelanoma miRNAome hsa-miR-3157-3p 711 1732 discovered in the breasttumor melanoma miRNAome hsa-miR-3157-5p 712 1733 discovered in thebreast tumor melanoma miRNAome hsa-miR-3158-3p 713 1734 discovered inthe breast tumor melanoma miRNAome, ovary hsa-miR-3158-5p 714 1735discovered in the breast tumor melanoma miRNAome, ovary hsa-miR-3159 7151736 discovered in the melanoma miRNAome hsa-miR-31-5p 716 1737 variouscancer cells (breast, lung, prostate) hsa-miR-3160-3p 717 1738discovered in the breast tumor melanoma miRNAome hsa-miR-3160-5p 7181739 discovered in the breast tumor melanoma miRNAome hsa-miR-3161 7191740 discovered in the melanoma miRNAome hsa-miR-3162-3p 720 1741discovered in the breast tumor melanoma miRNAome hsa-miR-3162-5p 7211742 discovered in the breast tumor melanoma miRNAome hsa-miR-3163 7221743 discovered in the melanoma miRNAome hsa-miR-3164 723 1744discovered in the melanoma miRNAome hsa-miR-3165 724 1745 discovered inthe breast tumor melanoma miRNAome hsa-miR-3166 725 1746 discovered inthe melanoma miRNAome hsa-miR-3167 726 1747 discovered in the melanomamiRNAome, ovary hsa-miR-3168 727 1748 discovered in the melanomamiRNAome hsa-miR-3169 728 1749 discovered in the melanoma miRNAomehsa-miR-3170 729 1750 discovered in the breast tumor melanoma miRNAomehsa-miR-3171 730 1751 discovered in the melanoma miRNAome, ovaryhsa-miR-3173-3p 731 1752 discovered in the breast tumor melanomamiRNAome hsa-miR-3173-5p 732 1753 discovered in the breast tumormelanoma miRNAome hsa-miR-3174 733 1754 discovered in the melanomamiRNAome hsa-miR-3175 734 1755 discovered in the breast tumor melanomamiRNAome, ovary hsa-miR-3176 735 1756 discovered in the breast tumormelanoma miRNAome hsa-miR-3177-3p 736 1757 discovered in the breasttumor and melanoma lymphoblastic miRNAome leukaemia hsa-miR-3177-5p 7371758 discovered in the breast tumor and melanoma lymphoblastic miRNAomeleukaemia hsa-miR-3178 738 1759 discovered in the melanoma miRNAomehsa-miR-3179 739 1760 discovered in the melanoma miRNAome hsa-miR-3180740 1761 discovered in the breast tumor melanoma miRNAome, ovaryhsa-miR-3180-3p 741 1762 discovered in breast tunor hsa-miR-3180-5p 7421763 discovered in breast tumor hsa-miR-3181 743 1764 discovered in themelanoma miRNAome hsa-miR-3182 744 1765 discovered in the melanomamiRNAome hsa-miR-3183 745 1766 discovered in the melanoma miRNAomehsa-miR-3184-3p 746 1767 discovered in the melanoma miRNAomehsa-miR-3184-5p 747 1768 discovered in the melanoma miRNAomehsa-miR-3185 748 1769 discovered in the melanoma miRNAomehsa-miR-3186-3p 749 1770 discovered in the melanoma miRNAome, ovaryhsa-miR-3186-5p 750 1771 discovered in the melanoma miRNAome, ovaryhsa-miR-3187-3p 751 1772 discovered in the breast tumor melanomamiRNAome hsa-miR-3187-5p 752 1773 discovered in the breast tumormelanoma miRNAome hsa-miR-3188 753 1774 discovered in the melanomamiRNAome hsa-miR-3189-3p 754 1775 discovered in the breast tumormelanoma miRNAome hsa-miR-3189-5p 755 1776 discovered in the breasttumor melanoma miRNAome hsa-miR-3190-3p 756 1777 discovered in thelymphoblastic melanoma leukaemia miRNAome hsa-miR-3190-5p 757 1778discovered in the lymphoblastic melanoma leukaemia miRNAomehsa-miR-3191-3p 758 1779 discovered in the melanoma miRNAomehsa-miR-3191-5p 759 1780 discovered in the melanoma miRNAomehsa-miR-3192 760 1781 discovered in the breast tumor melanoma miRNAomehsa-miR-3193 761 1782 discovered in the melanoma miRNAomehsa-miR-3194-3p 762 1783 discovered in the breast tumor melanomamiRNAome hsa-miR-3194-5p 763 1784 discovered in the breast tumormelanoma miRNAome hsa-miR-3195 764 1785 discovered in the melanomamiRNAome hsa-miR-3196 765 1786 basal cell carcinoma hsa-miR-3197 7661787 discovered in the melanoma miRNAome hsa-miR-3198 767 1788discovered in the breast tumor melanoma miRNAome hsa-miR-3199 768 1789discovered in the melanoma miRNAome hsa-miR-3200-3p 769 1790 discoveredin the breast tumor melanoma miRNAome, ovary hsa-miR-3200-5p 770 1791discovered in the breast tumor melanoma miRNAome, ovary hsa-miR-3201 7711792 discovered in the melanoma miRNAome, hsa-miR-3202 772 1793discovered in the melanoma miRNAome, epithelial cell BEAS2B hsa-miR-320a773 1794 blood, colon cancer cells, heart(myocardiac) heart diseasehsa-miR-320b 774 1795 central nevous system hsa-miR-320c 775 1796chondrocyte cartilage metabolism hsa-miR-320d 776 1797 cancer stem cellshsa-miR-320e 777 1798 neural cells hsa-miR-323a-3p 778 1799 neuronsmyeloid leukaemia, mudulla thyroid carcinoma hsa-miR-323a-5p 779 1800neurons myeloid leukaemia, mudulla thyroid carcinoma hsa-miR-323b-3p 7801801 myeloid leukaemia hsa-miR-323b-5p 781 1802 myeloid leukaemiahsa-miR-32-3p 782 1803 blood, glia various cancers (lung, kidney,prostate, etc), virus infection hsa-miR-324-3p 783 1804 kidneyhsa-miR-324-5p 784 1805 neurons tumor cells hsa-miR-325 785 1806neurons, placenta hsa-miR-32-5p 786 1807 blood, glia various cancers(lung, kidney, prostate, etc), virus infection hsa-miR-326 787 1808neurons tumor cells hsa-miR-328 788 1809 neuron, blood tumor cellshsa-miR-329 789 1810 brain and platele hsa-miR-330-3p 790 1811 variouscancers (prostate, glioblastoma, colorectal) hsa-miR-330-5p 791 1812various cancers (prostate, glioblastoma, colorectal) hsa-miR-331-3p 7921813 gastric cancer hsa-miR-331-5p 793 1814 lymphocytes hsa-miR-335-3p794 1815 kidney, breast RCC, multiple myeloma hsa-miR-335-5p 795 1816kidney, breast RCC, multiple myeloma hsa-miR-337-3p 796 1817 lunggastric cancer hsa-miR-337-5p 797 1818 lung hsa-miR-338-3p 798 1819epithelial cells, gastric, rectal oligodendrocytes cancer cells,osteosarcoma hsa-miR-338-5p 799 1820 oligodendrocytes gastric cancerhsa-miR-339-3p 800 1821 immune cell hsa-miR-339-5p 801 1822 immune cellhsa-miR-33a-3p 802 1823 pancreatic islet, lipid metabolism lipidmetabolism hsa-miR-33a-5p 803 1824 pancreatic islet, lipid metabolismlipid metabolism hsa-miR-33b-3p 804 1825 lipid metabolism lipidmetabolism hsa-miR-33b-5p 805 1826 lipid metabolism lipid metabolismhsa-miR-340-3p 806 1827 various cancers hsa-miR-340-5p 807 1828 embryoidbody cells hsa-miR-342-3p 808 1829 brain, circulating multiple myeloma,plasma other cancers hsa-miR-342-5p 809 1830 circulating plasma multiplemyeloma, other cancers hsa-miR-345-3p 810 1831 hematopoietic follicularcells lymphoma, other cancers hsa-miR-345-5p 811 1832 hematopoieticfollicular cells lymphoma, other cancers hsa-miR-346 812 1833 immumecells cancers and autoimmune hsa-miR-34a-3p 813 1834 breast, meyloidgastric cancer, tumor cells, ciliated CLL, other suppressor, p53epithelial cells inducible hsa-miR-34a-5p 814 1835 breast, meyloidgastric cancer, tumor cells, ciliated CLL, other suppressor, p53epithelial cells inducible hsa-miR-34b-3p 815 1836 ciliated epithelialvarious cancers tumor cells suppressor, p53 inducible hsa-miR-34b-5p 8161837 ciliated epithelial various cancers tumor cells suppressor, p53inducible hsa-miR-34c-3p 817 1838 ciliated epithelial various cancerstumor cells, placenta suppressor, p53 inducible hsa-miR-34c-5p 818 1839ciliated epithelial various cancers tumor cells, placenta suppressor,p53 inducible hsa-miR-3529-3p 819 1840 discovered in breast tumorhsa-miR-3529-5p 820 1841 discovered in breast tumor hsa-miR-3591-3p 8211842 discovered in breast tumor hsa-miR-3591-5p 822 1843 discovered inbreast tumor hsa-miR-3605-3p 823 1844 discovered in reprodcutive tractshsa-miR-3605-5p 824 1845 discovered in reprodcutive tractshsa-miR-3606-3p 825 1846 discovered in cervical tumors hsa-miR-3606-5p826 1847 discovered in cervical tumors hsa-miR-3607-3p 827 1848discovered in cervical tumors hsa-miR-3607-5p 828 1849 discovered incervical tumors hsa-miR-3609 829 1850 discovered in cervical tumorshsa-miR-3610 830 1851 discovered in cervical tumors hsa-miR-3611 8311852 discovered in cervical tumors hsa-miR-3612 832 1853 discovered incervical tumors hsa-miR-3613-3p 833 1854 discovered in cervical tumorshsa-miR-3613-5p 834 1855 discovered in cervical tumors hsa-miR-361-3p835 1856 blood, endothelial cells hsa-miR-3614-3p 836 1857 discovered incervical and breast tumors hsa-miR-3614-5p 837 1858 discovered incervical and breast tumors hsa-miR-3615 838 1859 discovered in cervicaltumors hsa-miR-361-5p 839 1860 endothelial cells hsa-miR-3616-3p 8401861 discovered in cervical tumors hsa-miR-3616-5p 841 1862 discoveredin cervical tumors hsa-miR-3617-3p 842 1863 discovered in cervicaltumors and psoriasis hsa-miR-3617-5p 843 1864 discovered in cervicaltumors and psoriasis hsa-miR-3618 844 1865 discovered in cervical tumorshsa-miR-3619-3p 845 1866 discovered in breast tumors hsa-miR-3619-5p 8461867 discovered in breast tumors hsa-miR-3620-3p 847 1868 discovered incervical tumors hsa-miR-3620-5p 848 1869 discovered in cervical tumorshsa-miR-3621 849 1870 discovered in cervical tumors hsa-miR-3622a-3p 8501871 discovered in breast tumors hsa-miR-3622a-5p 851 1872 discovered inbreast tumors hsa-miR-3622b-3p 852 1873 discovered in cervical tumorshsa-miR-3622b-5p 853 1874 discovered in cervical tumors hsa-miR-362-3p854 1875 melanoma hsa-miR-362-5p 855 1876 melanoma hsa-miR-363-3p 8561877 kidney stem cell, blood cells hsa-miR-363-5p 857 1878 kidney stemcell, blood cells hsa-miR-3646 858 1879 discovered in solid tumorhsa-miR-3648 859 1880 discovered in solid tumor hsa-miR-3649 860 1881discovered in solid tumor hsa-miR-3650 861 1882 discovered in solidtumor hsa-miR-3651 862 1883 discovered in solid tumor hsa-miR-3652 8631884 discovered in solid tumor hsa-miR-3653 864 1885 discovered in solidtumor hsa-miR-3654 865 1886 discovered in solid tumor hsa-miR-3655 8661887 discovered in solid tumor hsa-miR-3656 867 1888 discovered in solidtumor hsa-miR-3657 868 1889 discovered in solid tumor hsa-miR-3658 8691890 discovered in solid tumor hsa-miR-3659 870 1891 discovered inbreast tumors hsa-miR-365a-3p 871 1892 various cancer apoptosis cells(Immune cells, lung, colon, endometriotic) hsa-miR-365a-5p 872 1893various cancer apoptosis cells (Immune cells, lung, colon,endometriotic)) hsa-miR-365b-3p 873 1894 various cancers apoptosis(retinoblastoma, colon, endometriotic) hsa-miR-365b-5p 874 1895 variouscancers apoptosis (colon, endometriotic) hsa-miR-3660 875 1896discovered in breast tumors hsa-miR-3661 876 1897 discovered in breasttumors hsa-miR-3662 877 1898 — hsa-miR-3663-3p 878 1899 —hsa-miR-3663-5p 879 1900 — hsa-miR-3664-3p 880 1901 discovered in breasttumors hsa-miR-3664-5p 881 1902 discovered in breast tumors hsa-miR-3665882 1903 brain hsa-miR-3666 883 1904 brain hsa-miR-3667-3p 884 1905discovered in peripheral blood hsa-miR-3667-5p 885 1906 discovered inperipheral blood hsa-miR-3668 886 1907 discovered in peripheral bloodhsa-miR-3669 887 1908 discovered in peripheral blood hsa-miR-3670 8881909 discovered in peripheral blood hsa-miR-3671 889 1910 discovered inperipheral blood hsa-miR-3672 890 1911 discovered in peripheral bloodhsa-miR-3673 891 1912 discovered in peripheral blood hsa-miR-367-3p 8921913 embryonic stem reprogramming cells hsa-miR-3674 893 1914 discoveredin peripheral blood hsa-miR-3675-3p 894 1915 discovered in peripheralblood hsa-miR-3675-5p 895 1916 discovered in peripheral bloodhsa-miR-367-5p 896 1917 embryonic stem reprogramming cellshsa-miR-3676-3p 897 1918 discovered in peripheral blood hsa-miR-3676-5p898 1919 discovered in peripheral blood hsa-miR-3677-3p 899 1920discovered in peripheral blood hsa-miR-3677-5p 900 1921 discovered inperipheral blood hsa-miR-3678-3p 901 1922 discovered in peripheral bloodhsa-miR-3678-5p 902 1923 discovered in peripheral blood hsa-miR-3679-3p903 1924 discovered in peripheral blood hsa-miR-3679-5p 904 1925discovered in peripheral blood hsa-miR-3680-3p 905 1926 discovered inperipheral blood hsa-miR-3680-5p 906 1927 discovered in peripheral bloodhsa-miR-3681-3p 907 1928 discovered in peripheral blood hsa-miR-3681-5p908 1929 discovered in peripheral blood hsa-miR-3682-3p 909 1930discovered in peripheral blood hsa-miR-3682-5p 910 1931 discovered inperipheral blood hsa-miR-3683 911 1932 discovered in peripheral bloodhsa-miR-3684 912 1933 discovered in peripheral blood hsa-miR-3685 9131934 discovered in peripheral blood hsa-miR-3686 914 1935 discovered inperipheral blood hsa-miR-3687 915 1936 discovered in peripheral bloodhsa-miR-3688-3p 916 1937 discovered in breast tumor hsa-miR-3688-5p 9171938 discovered in breast tumor hsa-miR-3689a-3p 918 1939 discovered infemale reproductuve tract hsa-miR-3689a-5p 919 1940 discovered in femalereproductuve tract and peripheral blood hsa-miR-3689b-3p 920 1941discovered in female reproductuve tract and peripheral bloodhsa-miR-3689b-5p 921 1942 discovered in female reproductuve tracthsa-miR-3689c 922 1943 discovered in B cells hsa-miR-3689d 923 1944discovered in B cells hsa-miR-3689e 924 1945 discovered in B cellshsa-miR-3689f 925 1946 discovered in B cells hsa-miR-3690 926 1947discovered in peripheral blood hsa-miR-3691-3p 927 1948 discovered inperipheral blood hsa-miR-3691-5p 928 1949 discovered in peripheral bloodhsa-miR-3692-3p 929 1950 discovered in peripheral blood hsa-miR-3692-5p930 1951 discovered in peripheral blood hsa-miR-369-3p 931 1952 stemcells reprogramming hsa-miR-369-5p 932 1953 stem cells reprogramminghsa-miR-370 933 1954 acute meyloid tumor leukaemia and suppressor, lipidother cancers metabolism hsa-miR-3713 934 1955 discovered inneuroblastoma hsa-miR-3714 935 1956 discovered in neuroblastomahsa-miR-371a-3p 936 1957 serum hsa-miR-371a-5p 937 1958 serumhsa-miR-371b-3p 938 1959 serum hsa-miR-371b-5p 939 1960 serumhsa-miR-372 940 1961 hematopoietic cells, lung, placental (blood)hsa-miR-373-3p 941 1962 breast cancer hsa-miR-373-5p 942 1963 breastcancer hsa-miR-374a-3p 943 1964 muscle breast and lung myogenic(myoblasts) cancer differentiation hsa-miR-374a-5p 944 1965 musclebreast and lung myogenic (myoblasts) cancer differentiationhsa-miR-374b-3p 945 1966 muscle myogenic (myoblasts) differentiationhsa-miR-374b-5p 946 1967 muscle myogenic (myoblasts) differentiationhsa-miR-374c-3p 947 1968 muscle myogenic (myoblasts) differentiationhsa-miR-374c-5p 948 1969 muscle myogenic (myoblasts) differentiationhsa-miR-375 949 1970 pancreas (islet) hsa-miR-376a-2-5p 950 1971regulatory miRs for hematopoietic cells (erythroid, platelet, lympho)hsa-miR-376a-3p 951 1972 regulatory miRs for hematopoietic cells(erythroid, platelet, lympho) hsa-miR-376a-5p 952 1973 regulatory miRsfor hematopoietic cells (erythroid, platelet, lympho) hsa-miR-376b-3p953 1974 blood various cancer autophagy cells hsa-miR-376b-5p 954 1975blood various cancer autophagy cells hsa-miR-376c-3p 955 1976trophoblast various cancer cell proliferatio cells hsa-miR-376c-5p 9561977 trophoblast various cancer cell proliferatio cells hsa-miR-377-3p957 1978 hematopoietic cells hsa-miR-377-5p 958 1979 hematopoietic cellshsa-miR-378a-3p 959 1980 ovary, lipid metabolism hsa-miR-378a-5p 9601981 ovary, placenta/trophoblast, lipid metabolism hsa-miR-378b 961 1982lipid metabolism hsa-miR-378c 962 1983 lipid metabolism hsa-miR-378d 9631984 lipid metabolism hsa-miR-378e 964 1985 lipid metabolismhsa-miR-378f 965 1986 lipid metabolism hsa-miR-378g 966 1987 lipidmetabolism hsa-miR-378h 967 1988 lipid metabolism hsa-miR-378i 968 1989lipid metabolism hsa-miR-378j 969 1990 lipid metabolism hsa-miR-379-3p970 1991 various cancers (breast, hepatocytes, colon) hsa-miR-379-5p 9711992 various cancers (breast, hepatocytes, colon) hsa-miR-380-3p 9721993 brain neuroblastoma hsa-miR-380-5p 973 1994 brain, embryonicneuroblastoma stem cells hsa-miR-381-3p 974 1995 chondrogenesis, lung,brain hsa-miR-381-5p 975 1996 chondrogenesis, lung, brain hsa-miR-382-3p976 1997 renal epithelial cells hsa-miR-382-5p 977 1998 renal epithelialcells hsa-miR-383 978 1999 testes, brain (medulla) hsa-miR-384 979 2000epithelial cells hsa-miR-3907 980 2001 discovered in female reproductivetract hsa-miR-3908 981 2002 discovered in female reproductive tracthsa-miR-3909 982 2003 discovered in female reproductive tracthsa-miR-3910 983 2004 discovered in female reproductive tracthsa-miR-3911 984 2005 discovered in breast tumor and female reproductivetract hsa-miR-3912 985 2006 discovered in female reproductive tracthsa-miR-3913-3p 986 2007 discovered in breast tumor and femalereproductive tract hsa-miR-3913-5p 987 2008 discovered in breast tumorand female reproductive tract hsa-miR-3914 988 2009 discovered in breasttumor and female reproductive tract hsa-miR-3915 989 2010 discovered infemale reproductive tract hsa-miR-3916 990 2011 discovered in femalereproductive tract hsa-miR-3917 991 2012 discovered in femalereproductive tract hsa-miR-3918 992 2013 discovered in femalereproductive tract hsa-miR-3919 993 2014 discovered in femalereproductive tract hsa-miR-3920 994 2015 discovered in femalereproductive tract hsa-miR-3921 995 2016 discovered in femalereproductive tract hsa-miR-3922-3p 996 2017 discovered in breast tumorand female reproductive tract hsa-miR-3922-5p 997 2018 discovered inbreast tumor and female reproductive tract hsa-miR-3923 998 2019discovered in female reproductive tract hsa-miR-3924 999 2020 discoveredin female reproductive tract hsa-miR-3925-3p 1000 2021 discovered inbreast tumor and female reproductive tract hsa-miR-3925-5p 1001 2022discovered in breast tumor and female reproductive tract hsa-miR-39261002 2023 discovered in female reproductive tract hsa-miR-3927-3p 10032024 discovered in female reproductive tract and psoriasishsa-miR-3927-5p 1004 2025 discovered in female reproductive tract andpsoriasis hsa-miR-3928 1005 2026 discovered in female reproductive tracthsa-miR-3929 1006 2027 discovered in female reproductive tracthsa-miR-3934-3p 1007 2028 discovered in abnormal skin (psoriasis)hsa-miR-3934-5p 1008 2029 discovered in abnormal skin (psoriasis)hsa-miR-3935 1009 2030 hsa-miR-3936 1010 2031 discovered in breast tumorand lymphoblastic leukaemia hsa-miR-3937 1011 2032 hsa-miR-3938 10122033 hsa-miR-3939 1013 2034 hsa-miR-3940-3p 1014 2035 discovered inbreast tumor hsa-miR-3940-5p 1015 2036 discovered in breast tumorhsa-miR-3941 1016 2037 hsa-miR-3942-3p 1017 2038 discovered in breasttumor and lymphoblastic leukaemia hsa-miR-3942-5p 1018 2039 discoveredin breast tumor and lymphoblastic leukaemia hsa-miR-3943 1019 2040hsa-miR-3944-3p 1020 2041 discovered in breast tumor hsa-miR-3944-5p1021 2042 discovered in breast tumor hsa-miR-3945 1022 2043 hsa-miR-39601023 2044 osteoblast hsa-miR-3972 1024 2045 discovered in Acute MyeloidLeukaemia hsa-miR-3973 1025 2046 discovered in Acute Myeloid Leukaemiahsa-miR-3974 1026 2047 discovered in Acute Myeloid Leukaemiahsa-miR-3975 1027 2048 discovered in Acute Myeloid Leukaemiahsa-miR-3976 1028 2049 discovered in Acute Myeloid Leukaemiahsa-miR-3977 1029 2050 discovered in Acute Myeloid Leukaemiahsa-miR-3978 1030 2051 discovered in Acute Myeloid Leukaemiahsa-miR-409-3p 1031 2052 gastric cancer hsa-miR-409-5p 1032 2053 gastriccancer hsa-miR-410 1033 2054 brain glioma hsa-miR-411-3p 1034 2055Glioblastoma others hsa-miR-411-5p 1035 2056 Glioblastoma othershsa-miR-412 1036 2057 upregulated in lung cancer hsa-miR-421 1037 2058endothelial cells gastric cancer, HCC hsa-miR-422a 1038 2059 circulatingmicroRNA (in plasma) hsa-miR-423-3p 1039 2060 embryonic stem cellshsa-miR-423-5p 1040 2061 heart, embryonic stem cells hsa-miR-424-3p 10412062 endothelial cells various pro-angiogenic cancers(e.g B- lieageALL), cardiac diseases hsa-miR-424-5p 1042 2063 endothelial cellsvarious pro-angiogenic cancers(e.g B- lieage ALL), cardiac diseaseshsa-miR-4251 1043 2064 discovered in embryonic stem cells and neuralprecusors hsa-miR-4252 1044 2065 discovered in embryonic stem cells andneural precusors hsa-miR-4253 1045 2066 discovered in embryonic stemcells and neural precusors hsa-miR-425-3p 1046 2067 brain ovariancancer, brain tumor hsa-miR-4254 1047 2068 discovered in embryonic stemcells and neural precusors hsa-miR-4255 1048 2069 discovered inembryonic stem cells and neural precusors hsa-miR-425-5p 1049 2070 brainB-lieage ALL, brain tumor hsa-miR-4256 1050 2071 discovered in embryonicstem cells and neural precusors hsa-miR-4257 1051 2072 discovered inembryonic stem cells and neural precusors hsa-miR-4258 1052 2073discovered in embryonic stem cells and neural precusors hsa-miR-42591053 2074 discovered in embryonic stem cells and neural precusorshsa-miR-4260 1054 2075 discovered in embryonic stem cells and neuralprecusors hsa-miR-4261 1055 2076 discovered in embryonic stem cells andneural precusors hsa-miR-4262 1056 2077 discovered in embryonic stemcells and neural precusors hsa-miR-4263 1057 2078 discovered inembryonic stem cells and neural precusors hsa-miR-4264 1058 2079discovered in embryonic stem cells and neural precusors hsa-miR-42651059 2080 discovered in embryonic stem cells and neural precusorshsa-miR-4266 1060 2081 discovered in embryonic stem cells and neuralprecusors hsa-miR-4267 1061 2082 discovered in embryonic stem cells andneural precusors hsa-miR-4268 1062 2083 discovered in embryonic stemcells and neural precusors hsa-miR-4269 1063 2084 discovered inembryonic stem cells and neural precusors hsa-miR-4270 1064 2085discovered in embryonic stem cells and neural precusors hsa-miR-42711065 2086 discovered in embryonic stem cells and neural precusorshsa-miR-4272 1066 2087 discovered in embryonic stem cells and neuralprecusors hsa-miR-4273 1067 2088 hsa-miR-4274 1068 2089 discovered inembryonic stem cells and neural precusors hsa-miR-4275 1069 2090discovered in embryonic stem cells and neural precusors hsa-miR-42761070 2091 discovered in embryonic stem cells and neural precusorshsa-miR-4277 1071 2092 discovered in embryonic stem cells and neuralprecusors hsa-miR-4278 1072 2093 discovered in embryonic stem cells andneural precusors hsa-miR-4279 1073 2094 discovered in embryonic stemcells and neural precusors hsa-miR-4280 1074 2095 discovered inembryonic stem cells and neural precusors hsa-miR-4281 1075 2096discovered in embryonic stem cells and neural precusors hsa-miR-42821076 2097 discovered in embryonic stem cells and neural precusorshsa-miR-4283 1077 2098 discovered in embryonic stem cells and neuralprecusors hsa-miR-4284 1078 2099 discovered in embryonic stem cells andneural precusors hsa-miR-4285 1079 2100 discovered in embryonic stemcells and neural precusors hsa-miR-4286 1080 2101 discovered inembryonic stem cells and neural precusors hsa-miR-4287 1081 2102discovered in embryonic stem cells and neural precusors hsa-miR-42881082 2103 discovered in embryonic stem cells and neural precusorshsa-miR-4289 1083 2104 discovered in embryonic stem cells and neuralprecusors hsa-miR-429 1084 2105 Epithelial cells various cancers(colorectal, endometrial, gastric, ovarian etc) hsa-miR-4290 1085 2106discovered in embryonic stem cells and neural precusors hsa-miR-42911086 2107 discovered in embryonic stem cells and neural precusorshsa-miR-4292 1087 2108 discovered in embryonic stem cells and neuralprecusors hsa-miR-4293 1088 2109 discovered in embryonic stem cells andneural precusors hsa-miR-4294 1089 2110 discovered in embryonic stemcells and neural precusors hsa-miR-4295 1090 2111 discovered inembryonic stem cells and neural precusors hsa-miR-4296 1091 2112discovered in embryonic stem cells and neural precusors hsa-miR-42971092 2113 discovered in embryonic stem cells and neural precusorshsa-miR-4298 1093 2114 discovered in embryonic stem cells and neuralprecusors hsa-miR-4299 1094 2115 discovered in embryonic stem cells andneural precusors hsa-miR-4300 1095 2116 discovered in embryonic stemcells and neural precusors hsa-miR-4301 1096 2117 discovered inembryonic stem cells and neural precusors hsa-miR-4302 1097 2118discovered in embryonic stem cells and neural precusors hsa-miR-43031098 2119 discovered in embryonic stem cells and neural precusorshsa-miR-4304 1099 2120 discovered in embryonic stem cells and neuralprecusors hsa-miR-4305 1100 2121 discovered in embryonic stem cells andneural precusors hsa-miR-4306 1101 2122 discovered in embryonic stemcells and neural precusors hsa-miR-4307 1102 2123 discovered inembryonic stem cells and neural precusors hsa-miR-4308 1103 2124discovered in embryonic stem cells and neural precusors hsa-miR-43091104 2125 discovered in embryonic stem cells and neural precusorshsa-miR-4310 1105 2126 discovered in embryonic stem cells and neuralprecusors hsa-miR-4311 1106 2127 discovered in embryonic stem cells andneural precusors hsa-miR-4312 1107 2128 discovered in embryonic stemcells and neural precusors hsa-miR-4313 1108 2129 discovered inembryonic stem cells and neural precusors hsa-miR-431-3p 1109 2130Cancers (follicular lymphoma) hsa-miR-4314 1110 2131 discovered inembryonic stem cells and neural precusors hsa-miR-4315 1111 2132discovered in embryonic stem cells and neural precusors hsa-miR-431-5p1112 2133 Cancers (follicular lymphoma) hsa-miR-4316 1113 2134discovered in embryonic stem cells and neural precusors hsa-miR-43171114 2135 discovered in embryonic stem cells and neural precusorshsa-miR-4318 1115 2136 discovered in embryonic stem cells and neuralprecusors hsa-miR-4319 1116 2137 discovered in embryonic stem cells andneural precusors hsa-miR-4320 1117 2138 discovered in embryonic stemcells and neural precusors hsa-miR-4321 1118 2139 discovered inembryonic stem cells and neural precusors hsa-miR-4322 1119 2140discovered in embryonic stem cells and neural precusors hsa-miR-43231120 2141 discovered in embryonic stem cells and neural precusorshsa-miR-432-3p 1121 2142 myoblast myogenic differentiation hsa-miR-43241122 2143 discovered in embryonic stem cells and neural precusorshsa-miR-4325 1123 2144 discovered in embryonic stem cells and neuralprecusors hsa-miR-432-5p 1124 2145 myoblast myogenic differentiationhsa-miR-4326 1125 2146 discovered in embryonic stem cells and neuralprecusors hsa-miR-4327 1126 2147 discovered in embryonic stem cells andneural precusors hsa-miR-4328 1127 2148 discovered in embryonic stemcells and neural precusors hsa-miR-4329 1128 2149 discovered inembryonic stem cells and neural precusors hsa-miR-433 1129 2150 variousdiseases (cancer, Parkinson's, Chondrodysplasia) hsa-miR-4330 1130 2151discovered in embryonic stem cells and neural precusors hsa-miR-44171131 2152 discovered in B cells hsa-miR-4418 1132 2153 discovered in Bcells hsa-miR-4419a 1133 2154 discovered in B cells hsa-miR-4419b 11342155 discovered in B cells hsa-miR-4420 1135 2156 discovered in B cellshsa-miR-4421 1136 2157 discovered in B cells hsa-miR-4422 1137 2158discovered in breast tumor and B cells hsa-miR-4423-3p 1138 2159discovered in breast tumor, B cells and skin(psoriasis) hsa-miR-4423-5p1139 2160 discovered in breast tumor B cells and skin(psoriasis)hsa-miR-4424 1140 2161 discovered in B cells hsa-miR-4425 1141 2162discovered in B cells hsa-miR-4426 1142 2163 discovered in B cellshsa-miR-4427 1143 2164 discovered in B cells hsa-miR-4428 1144 2165discovered in B cells hsa-miR-4429 1145 2166 discovered in B cellshsa-miR-4430 1146 2167 discovered in B cells hsa-miR-4431 1147 2168discovered in B cells hsa-miR-4432 1148 2169 discovered in B cellshsa-miR-4433-3p 1149 2170 discovered in B cells hsa-miR-4433-5p 11502171 discovered in B cells hsa-miR-4434 1151 2172 discovered in B cellshsa-miR-4435 1152 2173 discovered in B cells hsa-miR-4436a 1153 2174discovered in breast tumor and B cells hsa-miR-4436b-3p 1154 2175discovered in breast tumor hsa-miR-4436b-5p 1155 2176 discovered inbreast tumor hsa-miR-4437 1156 2177 discovered in B cells hsa-miR-44381157 2178 discovered in B cells hsa-miR-4439 1158 2179 discovered in Bcells hsa-miR-4440 1159 2180 discovered in B cells hsa-miR-4441 11602181 discovered in B cells hsa-miR-4442 1161 2182 discovered in B cellshsa-miR-4443 1162 2183 discovered in B cells hsa-miR-4444 1163 2184discovered in B cells hsa-miR-4445-3p 1164 2185 discovered in B cellshsa-miR-4445-5p 1165 2186 discovered in B cells hsa-miR-4446-3p 11662187 discovered in breast tumor and B cells hsa-miR-4446-5p 1167 2188discovered in breast tumor and B cells hsa-miR-4447 1168 2189 discoveredin B cells hsa-miR-4448 1169 2190 discovered in B cells hsa-miR-44491170 2191 discovered in B cells hsa-miR-4450 1171 2192 discovered in Bcells hsa-miR-4451 1172 2193 discovered in B cells hsa-miR-4452 11732194 discovered in B cells hsa-miR-4453 1174 2195 discovered in B cellshsa-miR-4454 1175 2196 discovered in B cells hsa-miR-4455 1176 2197discovered in B cells hsa-miR-4456 1177 2198 discovered in B cellshsa-miR-4457 1178 2199 discovered in B cells hsa-miR-4458 1179 2200discovered in B cells hsa-miR-4459 1180 2201 discovered in B cellshsa-miR-4460 1181 2202 discovered in B cells hsa-miR-4461 1182 2203discovered in B cells hsa-miR-4462 1183 2204 discovered in B cellshsa-miR-4463 1184 2205 discovered in B cells hsa-miR-4464 1185 2206discovered in B cells hsa-miR-4465 1186 2207 discovered in B cellshsa-miR-4466 1187 2208 discovered in B cells hsa-miR-4467 1188 2209discovered in breast tumor and B cells hsa-miR-4468 1189 2210 discoveredin B cells hsa-miR-4469 1190 2211 discovered in breast tumor and B cellshsa-miR-4470 1191 2212 discovered in B cells hsa-miR-4471 2213 3234discovered in breast tumor and B cells hsa-miR-4472 2214 3235 discoveredin B cells hsa-miR-4473 2215 3236 discovered in B cells hsa-miR-4474-3p2216 3237 discovered in breast tumor, lymphoblastic leukaemia and Bcells hsa-miR-4474-5p 2217 3238 discovered in breast tumor,lymphoblastic leukaemia and B cells hsa-miR-4475 2218 3239 discovered inB cells hsa-miR-4476 2219 3240 discovered in B cells hsa-miR-4477a 22203241 discovered in B cells hsa-miR-4477b 2221 3242 discovered in B cellshsa-miR-4478 2222 3243 discovered in B cells hsa-miR-4479 2223 3244discovered in B cells hsa-miR-448 2224 3245 liver(hepatocytes) HCChsa-miR-4480 2225 3246 discovered in B cells hsa-miR-4481 2226 3247discovered in B cells hsa-miR-4482-3p 2227 3248 discovered in B cellshsa-miR-4482-5p 2228 3249 discovered in B cells hsa-miR-4483 2229 3250discovered in B cells hsa-miR-4484 2230 3251 discovered in B cellshsa-miR-4485 2231 3252 discovered in B cells hsa-miR-4486 2232 3253discovered in B cells hsa-miR-4487 2233 3254 discovered in B cellshsa-miR-4488 2234 3255 discovered in B cells hsa-miR-4489 2235 3256discovered in breast tumor and B cells hsa-miR-4490 2236 3257 discoveredin B cells hsa-miR-4491 2237 3258 discovered in B cells hsa-miR-44922238 3259 discovered in B cells hsa-miR-4493 2239 3260 discovered in Bcells hsa-miR-4494 2240 3261 discovered in B cells hsa-miR-4495 22413262 discovered in B cells hsa-miR-4496 2242 3263 discovered in B cellshsa-miR-4497 2243 3264 discovered in B cells hsa-miR-4498 2244 3265discovered in B cells hsa-miR-4499 2245 3266 discovered in B cellshsa-miR-449a 2246 3267 chondrocytes, ciliated lung, colonic, cell cycleepithelial cells ovarian cancer progression and proliferationhsa-miR-449b-3p 2247 3268 ciliated epithelial various cancer cell cyclecells, other tissues cells progression and proliferation hsa-miR-449b-5p2248 3269 ciliated epithelial various cancer cell cycle cells, othertissues cells progression and proliferation hsa-miR-449c-3p 2249 3270epithelial ovarian cancer cells hsa-miR-449c-5p 2250 3271 epithelialovarian cancer cells hsa-miR-4500 2251 3272 discovered in B cellshsa-miR-4501 2252 3273 discovered in B cells hsa-miR-4502 2253 3274discovered in B cells hsa-miR-4503 2254 3275 discovered in B cellshsa-miR-4504 2255 3276 discovered in B cells hsa-miR-4505 2256 3277discovered in B cells hsa-miR-4506 2257 3278 discovered in B cellshsa-miR-4507 2258 3279 discovered in B cells hsa-miR-4508 2259 3280discovered in B cells hsa-miR-4509 2260 3281 discovered in B cellshsa-miR-450a-3p 2261 3282 hsa-miR-450a-5p 2262 3283 hsa-miR-450b-3p 22633284 hsa-miR-450b-5p 2264 3285 hsa-miR-4510 2265 3286 discovered in Bcells hsa-miR-4511 2266 3287 discovered in B cells hsa-miR-4512 22673288 discovered in B cells hsa-miR-4513 2268 3289 discovered in B cellshsa-miR-4514 2269 3290 discovered in B cells hsa-miR-4515 2270 3291discovered in B cells hsa-miR-4516 2271 3292 discovered in B cellshsa-miR-4517 2272 3293 discovered in B cells hsa-miR-4518 2273 3294discovered in B cells hsa-miR-4519 2274 3295 discovered in B cellshsa-miR-451a 2275 3296 heart, central nevous system, epithelial cellshsa-miR-451b 2276 3297 heart, central nevous system, epithelial cellshsa-miR-4520a-3p 2277 3298 discovered in breast tumor and B cells,skin(psoriasis) hsa-miR-4520a-5p 2278 3299 discovered in breast tumorand B cells, skin(psoriasis) hsa-miR-4520b-3p 2279 3300 discovered inbreast tumor hsa-miR-4520b-5p 2280 3301 discovered in breast tumorhsa-miR-4521 2281 3302 discovered in B cells hsa-miR-4522 2282 3303discovered in B cells hsa-miR-4523 2283 3304 discovered in B cellshsa-miR-452-3p 2284 3305 myoblast bladder cancer and othershsa-miR-4524a-3p 2285 3306 discovered in breast tumor and B cells,skin(psoriasis) hsa-miR-4524a-5p 2286 3307 discovered in breast tumorand B cells, skin(psoriasis) hsa-miR-4524b-3p 2287 3308 discovered inbreast tumor and B cells, skin(psoriasis) hsa-miR-4524b-5p 2288 3309discovered in breast tumor and B cells, skin(psoriasis) hsa-miR-45252289 3310 discovered in B cells hsa-miR-452-5p 2290 3311 myoblastbladder cancer and others hsa-miR-4526 2291 3312 discovered in breasttumor and B cells hsa-miR-4527 2292 3313 discovered in B cellshsa-miR-4528 2293 3314 discovered in B cells hsa-miR-4529-3p 2294 3315discovered in breast tumor and B cells hsa-miR-4529-5p 2295 3316discovered in breast tumor and B cells hsa-miR-4530 2296 3317 discoveredin B cells hsa-miR-4531 2297 3318 discovered in B cells hsa-miR-45322298 3319 discovered in B cells hsa-miR-4533 2299 3320 discovered in Bcells hsa-miR-4534 2300 3321 discovered in B cells hsa-miR-4535 23013322 discovered in B cells hsa-miR-4536-3p 2302 3323 discovered in Bcells hsa-miR-4536-5p 2303 3324 discovered in B cells hsa-miR-4537 23043325 discovered in B cells hsa-miR-4538 2305 3326 discovered in B cellshsa-miR-4539 2306 3327 discovered in B cells hsa-miR-4540 2307 3328discovered in B cells hsa-miR-454-3p 2308 3329 embryoid body cells,central nevous system, monocytes hsa-miR-454-5p 2309 3330 embryoid bodycells, central nevous system, monocytes hsa-miR-455-3p 2310 3331 basalcell carcinoma, other cancers hsa-miR-455-5p 2311 3332 basal cellcarcinoma, other cancers hsa-miR-4632-3p 2312 3333 discovred in breasttumor hsa-miR-4632-5p 2313 3334 discovered in breast tumorhsa-miR-4633-3p 2314 3335 discovered in breast tumor hsa-miR-4633-5p2315 3336 discovered in breast tumor hsa-miR-4634 2316 3337 discoveredin breast tumor hsa-miR-4635 2317 3338 discovered in breast tumorhsa-miR-4636 2318 3339 discovered in breast tumor hsa-miR-4637 2319 3340discovered in breast tumor and lymphoblastic leukaemia hsa-miR-4638-3p2320 3341 discovered in breast tumor hsa-miR-4638-5p 2321 3342discovered in breast tumor hsa-miR-4639-3p 2322 3343 discovered inbreast tumor hsa-miR-4639-5p 2323 3344 discovered in breast tumorhsa-miR-4640-3p 2324 3345 discovered in breast tumor hsa-miR-4640-5p2325 3346 discovered in breast tumor hsa-miR-4641 2326 3347 discoveredin breast tumor hsa-miR-4642 2327 3348 discovered in breast tumorhsa-miR-4643 2328 3349 discovered in breast tumor hsa-miR-4644 2329 3350discovered in breast tumor hsa-miR-4645-3p 2330 3351 discovered inbreast tumor hsa-miR-4645-5p 2331 3352 discovered in breast tumorhsa-miR-4646-3p 2332 3353 discovered in breast tumor hsa-miR-4646-5p2333 3354 discovered in breast tumor hsa-miR-4647 2334 3355 discoveredin breast tumor hsa-miR-4648 2335 3356 discovered in breast tumorhsa-miR-4649-3p 2336 3357 discovered in breast tumor hsa-miR-4649-5p2337 3358 discovered in breast tumor hsa-miR-4650-3p 2338 3359discovered in breast tumor hsa-miR-4650-5p 2339 3360 discovered inbreast tumor hsa-miR-4651 2340 3361 discovered in breast tumorhsa-miR-4652-3p 2341 3362 discovered in breast tumor hsa-miR-4652-5p2342 3363 discovered in breast tumor hsa-miR-4653-3p 2343 3364discovered in breast tumor hsa-miR-4653-5p 2344 3365 discovered inbreast tumor hsa-miR-4654 2345 3366 discovered in breast tumorhsa-miR-4655-3p 2346 3367 discovered in breast tumor hsa-miR-4655-5p2347 3368 discovered in breast tumor hsa-miR-4656 2348 3369 discoveredin breast tumor hsa-miR-4657 2349 3370 discovered in breast tumorhsa-miR-4658 2350 3371 discovered in breast tumor hsa-miR-4659a-3p 23513372 discovered in breast tumor hsa-miR-4659a-5p 2352 3373 discovered inbreast tumor hsa-miR-4659b-3p 2353 3374 discovered in breast tumorhsa-miR-4659b-5p 2354 3375 discovered in breast tumor hsa-miR-466 23553376 hsa-miR-4660 2356 3377 discovered in breast tumor hsa-miR-4661-3p2357 3378 discovered in breast tumor hsa-miR-4661-5p 2358 3379discovered in breast tumor hsa-miR-4662a-3p 2359 3380 discovered inbreast tumor, psoriasis hsa-miR-4662a-5p 2360 3381 discovered in breasttumor, psoriasis hsa-miR-4662b 2361 3382 discovered in breast tumorhsa-miR-4663 2362 3383 discovered in breast tumor hsa-miR-4664-3p 23633384 discovered in breast tumor hsa-miR-4664-5p 2364 3385 discovered inbreast tumor hsa-miR-4665-3p 2365 3386 discovered in breast tumorhsa-miR-4665-5p 2366 3387 discovered in breast tumor hsa-miR-4666a-3p2367 3388 discovered in breast tumor hsa-miR-4666a-5p 2368 3389discovered in breast tumor hsa-miR-4666b 2369 3390 hsa-miR-4667-3p 23703391 discovered in breast tumor hsa-miR-4667-5p 2371 3392 discovered inbreast tumor hsa-miR-4668-3p 2372 3393 discovered in breast tumorhsa-miR-4668-5p 2373 3394 discovered in breast tumor hsa-miR-4669 23743395 discovered in breast tumor hsa-miR-4670-3p 2375 3396 discovered inbreast tumor hsa-miR-4670-5p 2376 3397 discovered in breast tumorhsa-miR-4671-3p 2377 3398 discovered in breast tumor hsa-miR-4671-5p2378 3399 discovered in breast tumor hsa-miR-4672 2379 3400 discoveredin breast tumor hsa-miR-4673 2380 3401 discovered in breast tumorhsa-miR-4674 2381 3402 discovered in breast tumor hsa-miR-4675 2382 3403discovered in breast tumor hsa-miR-4676-3p 2383 3404 discovered inbreast tumor hsa-miR-4676-5p 2384 3405 discovered in breast tumorhsa-miR-4677-3p 2385 3406 discovered in breast tumor, psoriasishsa-miR-4677-5p 2386 3407 discovered in breast tumor, psoriasishsa-miR-4678 2387 3408 discovered in breast tumor hsa-miR-4679 2388 3409discovered in breast tumor hsa-miR-4680-3p 2389 3410 discovered inbreast tumor hsa-miR-4680-5p 2390 3411 discovered in breast tumorhsa-miR-4681 2391 3412 discovered in breast tumor hsa-miR-4682 2392 3413discovered in breast tumor hsa-miR-4683 2393 3414 discovered in breasttumor hsa-miR-4684-3p 2394 3415 discovered in breast tumorhsa-miR-4684-5p 2395 3416 discovered in breast tumor hsa-miR-4685-3p2396 3417 discovered in breast tumor hsa-miR-4685-5p 2397 3418discovered in breast tumor hsa-miR-4686 2398 3419 discovered in breasttumor hsa-miR-4687-3p 2399 3420 discovered in breast tumorhsa-miR-4687-5p 2400 3421 discovered in breast tumor hsa-miR-4688 24013422 discovered in breast tumor hsa-miR-4689 2402 3423 discovered inbreast tumor hsa-miR-4690-3p 2403 3424 discovered in breast tumorhsa-miR-4690-5p 2404 3425 discovered in breast tumor hsa-miR-4691-3p2405 3426 discovered in breast tumor hsa-miR-4691-5p 2406 3427discovered in breast tumor hsa-miR-4692 2407 3428 discovered in breasttumor hsa-miR-4693-3p 2408 3429 discovered in breast tumorhsa-miR-4693-5p 2409 3430 discovered in breast tumor hsa-miR-4694-3p2410 3431 discovered in breast tumor hsa-miR-4694-5p 2411 3432discovered in breast tumor hsa-miR-4695-3p 2412 3433 discovered inbreast tumor hsa-miR-4695-5p 2413 3434 discovered in breast tumorhsa-miR-4696 2414 3435 discovered in breast tumor hsa-miR-4697-3p 24153436 discovered in breast tumor hsa-miR-4697-5p 2416 3437 discovered inbreast tumor hsa-miR-4698 2417 3438 discovered in breast tumorhsa-miR-4699-3p 2418 3439 discovered in breast tumor hsa-miR-4699-5p2419 3440 discovered in breast tumor hsa-miR-4700-3p 2420 3441discovered in breast tumor hsa-miR-4700-5p 2421 3442 discovered inbreast tumor hsa-miR-4701-3p 2422 3443 discovered in breast tumorhsa-miR-4701-5p 2423 3444 discovered in breast tumor hsa-miR-4703-3p2424 3445 discovered in breast tumor hsa-miR-4703-5p 2425 3446discovered in breast tumor hsa-miR-4704-3p 2426 3447 discovered inbreast tumor hsa-miR-4704-5p 2427 3448 discovered in breast tumorhsa-miR-4705 2428 3449 discovered in breast tumor hsa-miR-4706 2429 3450discovered in breast tumor hsa-miR-4707-3p 2430 3451 discovered inbreast tumor hsa-miR-4707-5p 2431 3452 discovered in breast tumorhsa-miR-4708-3p 2432 3453 discovered in breast tumor hsa-miR-4708-5p2433 3454 discovered in breast tumor hsa-miR-4709-3p 2434 3455discovered in breast tumor hsa-miR-4709-5p 2435 3456 discovered inbreast tumor hsa-miR-4710 2436 3457 discovered in breast tumorhsa-miR-4711-3p 2437 3458 discovered in breast tumor hsa-miR-4711-5p2438 3459 discovered in breast tumor hsa-miR-4712-3p 2439 3460discovered in breast tumor hsa-miR-4712-5p 2440 3461 discovered inbreast tumor hsa-miR-4713-3p 2441 3462 discovered in breast tumorhsa-miR-4713-5p 2442 3463 discovered in breast tumor hsa-miR-4714-3p2443 3464 discovered in breast tumor hsa-miR-4714-5p 2444 3465discovered in breast tumor hsa-miR-4715-3p 2445 3466 discovered inbreast tumor hsa-miR-4715-5p 2446 3467 discovered in breast tumorhsa-miR-4716-3p 2447 3468 discovered in breast tumor hsa-miR-4716-5p2448 3469 discovered in breast tumor hsa-miR-4717-3p 2449 3470discovered in breast tumor hsa-miR-4717-5p 2450 3471 discovered inbreast tumor hsa-miR-4718 2451 3472 discovered in breast tumorhsa-miR-4719 2452 3473 discovered in breast tumor hsa-miR-4720-3p 24533474 discovered in breast tumor hsa-miR-4720-5p 2454 3475 discovered inbreast tumor hsa-miR-4721 2455 3476 discovered in breast tumorhsa-miR-4722-3p 2456 3477 discovered in breast tumor hsa-miR-4722-5p2457 3478 discovered in breast tumor hsa-miR-4723-3p 2458 3479discovered in breast tumor hsa-miR-4723-5p 2459 3480 discovered inbreast tumor hsa-miR-4724-3p 2460 3481 discovered in breast tumorhsa-miR-4724-5p 2461 3482 discovered in breast tumor hsa-miR-4725-3p2462 3483 discovered in breast tumor hsa-miR-4725-5p 2463 3484discovered in breast tumor hsa-miR-4726-3p 2464 3485 discovered inbreast tumor hsa-miR-4726-5p 2465 3486 discovered in breast tumorhsa-miR-4727-3p 2466 3487 discovered in breast tumor hsa-miR-4727-5p2467 3488 discovered in breast tumor hsa-miR-4728-3p 2468 3489discovered in breast tumor hsa-miR-4728-5p 2469 3490 discovered inbreast tumor hsa-miR-4729 2470 3491 discovered in breast tumorhsa-miR-4730 2471 3492 discovered in breast tumor hsa-miR-4731-3p 24723493 discovered in breast tumor hsa-miR-4731-5p 2473 3494 discovered inbreast tumor hsa-miR-4732-3p 2474 3495 discovered in breast tumorhsa-miR-4732-5p 2475 3496 discovered in breast tumor hsa-miR-4733-3p2476 3497 discovered in breast tumor hsa-miR-4733-5p 2477 3498discovered in breast tumor hsa-miR-4734 2478 3499 discovered in breasttumor hsa-miR-4735-3p 2479 3500 discovered in breast tumorhsa-miR-4735-5p 2480 3501 discovered in breast tumor hsa-miR-4736 24813502 discovered in breast tumor hsa-miR-4737 2482 3503 discovered inbreast tumor hsa-miR-4738-3p 2483 3504 discovered in breast tumorhsa-miR-4738-5p 2484 3505 discovered in breast tumor hsa-miR-4739 24853506 discovered in breast tumor hsa-miR-4740-3p 2486 3507 discovered inbreast tumor hsa-miR-4740-5p 2487 3508 discovered in breast tumorhsa-miR-4741 2488 3509 discovered in breast tumor, psoriasishsa-miR-4742-3p 2489 3510 discovered in breast tumor, psoriasishsa-miR-4742-5p 2490 3511 discovered in breast tumor hsa-miR-4743-3p2491 3512 discovered in breast tumor hsa-miR-4743-5p 2492 3513discovered in breast tumor hsa-miR-4744 2493 3514 discovered in breasttumor hsa-miR-4745-3p 2494 3515 discovered in breast tumorhsa-miR-4745-5p 2495 3516 discovered in breast tumor hsa-miR-4746-3p2496 3517 discovered in breast tumor hsa-miR-4746-5p 2497 3518discovered in breast tumor hsa-miR-4747-3p 2498 3519 discovered inbreast tumor hsa-miR-4747-5p 2499 3520 discovered in breast tumorhsa-miR-4748 2500 3521 discovered in breast tumor hsa-miR-4749-3p 25013522 discovered in breast tumor hsa-miR-4749-5p 2502 3523 discovered inbreast tumor hsa-miR-4750-3p 2503 3524 discovered in breast tumorhsa-miR-4750-5p 2504 3525 discovered in breast tumor hsa-miR-4751 25053526 discovered in breast tumor hsa-miR-4752 2506 3527 discovered inbreast tumor hsa-miR-4753-3p 2507 3528 discovered in breast tumorhsa-miR-4753-5p 2508 3529 discovered in breast tumor hsa-miR-4754 25093530 discovered in breast tumor hsa-miR-4755-3p 2510 3531 discovered inbreast tumor hsa-miR-4755-5p 2511 3532 discovered in breast tumorhsa-miR-4756-3p 2512 3533 discovered in breast tumor hsa-miR-4756-5p2513 3534 discovered in breast tumor hsa-miR-4757-3p 2514 3535discovered in breast tumor hsa-miR-4757-5p 2515 3536 discovered inbreast tumor hsa-miR-4758-3p 2516 3537 discovered in breast tumorhsa-miR-4758-5p 2517 3538 discovered in breast tumor hsa-miR-4759 25183539 discovered in breast tumor hsa-miR-4760-3p 2519 3540 discovered inbreast tumor hsa-miR-4760-5p 2520 3541 discovered in breast tumorhsa-miR-4761-3p 2521 3542 discovered in breast tumor hsa-miR-4761-5p2522 3543 discovered in breast tumor hsa-miR-4762-3p 2523 3544discovered in breast tumor hsa-miR-4762-5p 2524 3545 discovered inbreast tumor hsa-miR-4763-3p 2525 3546 discovered in breast tumorhsa-miR-4763-5p 2526 3547 discovered in breast tumor hsa-miR-4764-3p2527 3548 discovered in breast tumor hsa-miR-4764-5p 2528 3549discovered in breast tumor hsa-miR-4765 2529 3550 discovered in breasttumor hsa-miR-4766-3p 2530 3551 discovered in breast tumorhsa-miR-4766-5p 2531 3552 discovered in breast tumor hsa-miR-4767 25323553 discovered in breast tumor hsa-miR-4768-3p 2533 3554 discovered inbreast tumor hsa-miR-4768-5p 2534 3555 discovered in breast tumorhsa-miR-4769-3p 2535 3556 discovered in breast tumor hsa-miR-4769-5p2536 3557 discovered in breast tumor hsa-miR-4770 2537 3558 discoveredin breast tumor hsa-miR-4771 2538 3559 discovered in breast tumorhsa-miR-4772-3p 2539 3560 discovered in energy breast tumor, metabolism/blood monoclear obesity cells hsa-miR-4772-5p 2540 3561 discovered inenergy breast tumor, metabolism/ blood monoclear obesity cellshsa-miR-4773 2541 3562 discovered in breast tumor hsa-miR-4774-3p 25423563 discovered in breast tumor and Lymphoblastic leukemiahsa-miR-4774-5p 2543 3564 discovered in breast tumor and Lymphoblasticleukemia hsa-miR-4775 2544 3565 discovered in breast tumorhsa-miR-4776-3p 2545 3566 discovered in breast tumor hsa-miR-4776-5p2546 3567 discovered in breast tumor hsa-miR-4777-3p 2547 3568discovered in breast tumor hsa-miR-4777-5p 2548 3569 discovered inbreast tumor hsa-miR-4778-3p 2549 3570 discovered in breast tumorhsa-miR-4778-5p 2550 3571 discovered in breast tumor hsa-miR-4779 25513572 discovered in breast tumor hsa-miR-4780 2552 3573 discovered inbreast tumor hsa-miR-4781-3p 2553 3574 discovered in breast tumorhsa-miR-4781-5p 2554 3575 discovered in breast tumor hsa-miR-4782-3p2555 3576 discovered in breast tumor hsa-miR-4782-5p 2556 3577discovered in breast tumor hsa-miR-4783-3p 2557 3578 discovered inbreast tumor hsa-miR-4783-5p 2558 3579 discovered in breast tumorhsa-miR-4784 2559 3580 discovered in breast tumor hsa-miR-4785 2560 3581discovered in breast tumor hsa-miR-4786-3p 2561 3582 discovered inbreast tumor hsa-miR-4786-5p 2562 3583 discovered in breast tumorhsa-miR-4787-3p 2563 3584 discovered in breast tumor hsa-miR-4787-5p2564 3585 discovered in breast tumor hsa-miR-4788 2565 3586 discoveredin breast tumor hsa-miR-4789-3p 2566 3587 discovered in breast tumorhsa-miR-4789-5p 2567 3588 discovered in breast tumor hsa-miR-4790-3p2568 3589 discovered in breast tumor hsa-miR-4790-5p 2569 3590discovered in breast tumor hsa-miR-4791 2570 3591 discovered in breasttumor hsa-miR-4792 2571 3592 discovered in breast tumor hsa-miR-4793-3p2572 3593 discovered in breast tumor hsa-miR-4793-5p 2573 3594discovered in breast tumor hsa-miR-4794 2574 3595 discovered in breasttumor hsa-miR-4795-3p 2575 3596 discovered in breast tumorhsa-miR-4795-5p 2576 3597 discovered in breast tumor hsa-miR-4796-3p2577 3598 discovered in breast tumor hsa-miR-4796-5p 2578 3599discovered in breast tumor hsa-miR-4797-3p 2579 3600 discovered inbreast tumor hsa-miR-4797-5p 2580 3601 discovered in breast tumorhsa-miR-4798-3p 2581 3602 discovered in breast tumor hsa-miR-4798-5p2582 3603 discovered in breast tumor hsa-miR-4799-3p 2583 3604discovered in breast tumor hsa-miR-4799-5p 2584 3605 discovered inbreast tumor hsa-miR-4800-3p 2585 3606 discovered in breast tumorhsa-miR-4800-5p 2586 3607 discovered in breast tumor hsa-miR-4801 25873608 discovered in breast tumor hsa-miR-4802-3p 2588 3609 discovered inbreast tumor, psoriasis hsa-miR-4802-5p 2589 3610 discovered in breasttumor, psoriasis hsa-miR-4803 2590 3611 discovered in breast tumorhsa-miR-4804-3p 2591 3612 discovered in breast tumor hsa-miR-4804-5p2592 3613 discovered in breast tumor hsa-miR-483-3p 2593 3614aderonocortical oncogenic carcinoma, rectal/pancreatic cancer,proliferation of wounded epithelial cells hsa-miR-483-5p 2594 3615cartilage aderonocortical angiogenesis (chondrocyte), carcinoma fetalbrain hsa-miR-484 2595 3616 mitochondrial network hsa-miR-485-3p 25963617 hsa-miR-485-5p 2597 3618 ovarian epithelial tumor hsa-miR-486-3p2598 3619 erythroid cells various cancers hsa-miR-486-5p 2599 3620 stemcells various cancers (adipose) hsa-miR-487a 2600 3621 laryngealcarcinoma hsa-miR-487b 2601 3622 neuroblastoma, pulmonary carcinogenesishsa-miR-488-3p 2602 3623 prostate cancer, others hsa-miR-488-5p 26033624 prostate cancer, others hsa-miR-489 2604 3625 mesenchymal stemosteogenesis cells hsa-miR-490-3p 2605 3626 neuroblastoma, terineleiomyoma (ULM)/muscle hsa-miR-490-5p 2606 3627 neuroblastoma, terineleiomyoma (ULM)/muscle hsa-miR-491-3p 2607 3628 various cancers,pro-apoptosis brain disease hsa-miR-491-5p 2608 3629 various cancers,pro-apoptosis brain disease hsa-miR-492 2609 3630 hsa-miR-493-3p 26103631 myeloid cells, pancreas (islet) hsa-miR-493-5p 2611 3632 myeloidcells, pancreas (islet) hsa-miR-494 2612 3633 epithelial cells variouscancers cell cycle hsa-miR-495-3p 2613 3634 platelet various cancers(gastric, MLL leukemia, pancreatic etc) and inflammation hsa-miR-495-5p2614 3635 platelet various cancers (gastric, MLL leukemia, pancreaticetc) and inflammation hsa-miR-496 2615 3636 Blood hsa-miR-497-3p 26163637 various cancers tumor (breast, colorectal, supressor/pro- etc)apoptosis hsa-miR-497-5p 2617 3638 various cancers tumor (breast,colorectal, supressor/pro- etc) apoptosis hsa-miR-498 2618 3639autoimmuno (e.g. rheumatoid arthritis) hsa-miR-4999-3p 2619 3640hsa-miR-4999-5p 2620 3641 hsa-miR-499a-3p 2621 3642 heart, cardiac stemcardiovascular cardiomyocyte cells disease differentiationhsa-miR-499a-5p 2622 3643 heart, cardiac stem cardiovascularcardiomyocyte cells disease differentiation hsa-miR-499b-3p 2623 3644heart, cardiac stem cardiovascular cardiomyocyte cells diseasedifferentiation hsa-miR-499b-5p 2624 3645 heart, cardiac stemcardiovascular cardiomyocyte cells disease differentiationhsa-miR-5000-3p 2625 3646 discovered in lymphoblastic leukaemiahsa-miR-5000-5p 2626 3647 discovered in lymphoblastic leukaemiahsa-miR-5001-3p 2627 3648 hsa-miR-5001-5p 2628 3649 hsa-miR-5002-3p 26293650 hsa-miR-5002-5p 2630 3651 hsa-miR-5003-3p 2631 3652 hsa-miR-5003-5p2632 3653 hsa-miR-5004-3p 2633 3654 hsa-miR-5004-5p 2634 3655hsa-miR-5006-3p 2635 3656 discovered in lymphoblastic leukaemiahsa-miR-5006-5p 2636 3657 discovered in lymphoblastic leukaemiahsa-miR-5007-3p 2637 3658 hsa-miR-5007-5p 2638 3659 hsa-miR-5008-3p 26393660 hsa-miR-5008-5p 2640 3661 hsa-miR-5009-3p 2641 3662 hsa-miR-5009-5p2642 3663 hsa-miR-500a-3p 2643 3664 hsa-miR-500a-5p 2644 3665hsa-miR-500b 2645 3666 Blood (plasma) hsa-miR-5010-3p 2646 3667 abnormalskin (psoriasis) hsa-miR-5010-5p 2647 3668 abnormal skin (psoriasis)hsa-miR-5011-3p 2648 3669 hsa-miR-5011-5p 2649 3670 hsa-miR-501-3p 26503671 hsa-miR-501-5p 2651 3672 hsa-miR-502-3p 2652 3673 various cancers(hepatocellular, ovarian, breast) hsa-miR-502-5p 2653 3674 variouscancers (hepatocellular, ovarian, breast) hsa-miR-503-3p 2654 3675 ovaryhsa-miR-503-5p 2655 3676 ovary hsa-miR-504 2656 3677 glioblastomahsa-miR-5047 2657 3678 hsa-miR-505-3p 2658 3679 breast cancerhsa-miR-505-5p 2659 3680 breast cancer hsa-miR-506-3p 2660 3681 variouscancers hsa-miR-506-5p 2661 3682 various cancers hsa-miR-507 2662 3683hsa-miR-508-3p 2663 3684 renal cell carcinoma hsa-miR-508-5p 2664 3685endothelial progenitor cells (EPCs) hsa-miR-5087 2665 3686 hsa-miR-50882666 3687 hsa-miR-5089-3p 2667 3688 hsa-miR-5089-5p 2668 3689hsa-miR-5090 2669 3690 hsa-miR-5091 2670 3691 hsa-miR-5092 2671 3692hsa-miR-5093 2672 3693 hsa-miR-509-3-5p 2673 3694 testis hsa-miR-509-3p2674 3695 renal cell carcinoma, brain disease hsa-miR-5094 2675 3696hsa-miR-5095 2676 3697 cervical cancer hsa-miR-509-5p 2677 3698metabolic syndrome, brain disease hsa-miR-5096 2678 3699 cervical cancehsa-miR-510 2679 3700 brain hsa-miR-5100 2680 3701 discoverd in Salivarygland hsa-miR-511 2681 3702 dendritic cells and macrophageshsa-miR-512-3p 2682 3703 embryonic stem cells, placenta hsa-miR-512-5p2683 3704 embryonic stem cells, placenta, hsa-miR-513a-3p 2684 3705 lungcarcinoma hsa-miR-513a-5p 2685 3706 endothelial cells hsa-miR-513b 26863707 follicular lymphoma hsa-miR-513c-3p 2687 3708 hsa-miR-513c-5p 26883709 hsa-miR-514a-3p 2689 3710 hsa-miR-514a-5p 2690 3711 hsa-miR-514b-3p2691 3712 various cancer cells hsa-miR-514b-5p 2692 3713 various cancercells hsa-miR-515-3p 2693 3714 hsa-miR-515-5p 2694 3715 placentahsa-miR-516a-3p 2695 3716 frontal cortex hsa-miR-516a-5p 2696 3717placenta hsa-miR-516b-3p 2697 3718 hsa-miR-516b-5p 2698 3719hsa-miR-517-5p 2699 3720 placenta hsa-miR-517a-3p 2700 3721 placentahsa-miR-517b-3p 2701 3722 placenta hsa-miR-517c-3p 2702 3723 placentahsa-miR-5186 2703 3724 discovered in lymphoblastic leukaemiahsa-miR-5187-3p 2704 3725 discovered in lymphoblastic leukaemia, skin(psoriasis) hsa-miR-5187-5p 2705 3726 discovered in lymphoblasticleukaemia, skin (psoriasis) hsa-miR-5188 2706 3727 discovered inlymphoblastic leukaemia hsa-miR-5189 2707 3728 discovered inlymphoblastic leukaemia hsa-miR-518a-3p 2708 3729 HCC hsa-miR-518a-5p2709 3730 various cancer cells hsa-miR-518b 2710 3731 placenta HCC cellcycle progression hsa-miR-518c-3p 2711 3732 placenta hsa-miR-518c-5p2712 3733 placenta hsa-miR-518d-3p 2713 3734 hsa-miR-518d-5p 2714 3735hsa-miR-518e-3p 2715 3736 HCC cell cycle progression hsa-miR-518e-5p2716 3737 HCC cell cycle progression hsa-miR-518f-3p 2717 3738 placentahsa-miR-518f-5p 2718 3739 placenta hsa-miR-5190 2719 3740 discovered inlymphoblastic leukaemia hsa-miR-5191 2720 3741 discovered inlymphoblastic leukaemia hsa-miR-5192 2721 3742 discovered inlymphoblastic leukaemia hsa-miR-5193 2722 3743 discovered inlymphoblastic leukaemia hsa-miR-5194 2723 3744 discovered inlymphoblastic leukaemia hsa-miR-5195-3p 2724 3745 discovered inlymphoblastic leukaemia hsa-miR-5195-5p 2725 3746 discovered inlymphoblastic leukaemia hsa-miR-5196-3p 2726 3747 discovered inlymphoblastic leukaemia hsa-miR-5196-5p 2727 3748 discovered inlymphoblastic leukaemia hsa-miR-5197-3p 2728 3749 discovered inlymphoblastic leukaemia hsa-miR-5197-5p 2729 3750 discovered inlymphoblastic leukaemia hsa-miR-519a-3p 2730 3751 placenta HCChsa-miR-519a-5p 2731 3752 placenta HCC hsa-miR-519b-3p 2732 3753 breastcancer hsa-miR-519b-5p 2733 3754 breast cancer hsa-miR-519c-3p 2734 3755hsa-miR-519c-5p 2735 3756 hsa-miR-519d 2736 3757 placentahsa-miR-519e-3p 2737 3758 placenta hsa-miR-519e-5p 2738 3759 placentahsa-miR-520a-3p 2739 3760 placenta hsa-miR-520a-5p 2740 3761 placentahsa-miR-520b 2741 3762 breast cancer hsa-miR-520c-3p 2742 3763 gastriccancer, breast tumor hsa-miR-520c-5p 2743 3764 breast tumorhsa-miR-520d-3p 2744 3765 various cancer cells hsa-miR-520d-5p 2745 3766various cancer cells hsa-miR-520e 2746 3767 hepatoma tomor suppressorhsa-miR-520f 2747 3768 breast cancer hsa-miR-520g 2748 3769 HCC, bladdercancer, breast cancer hsa-miR-520h 2749 3770 placental specifichsa-miR-521 2750 3771 prostate cancer hsa-miR-522-3p 2751 3772 HCChsa-miR-522-5p 2752 3773 HCC hsa-miR-523-3p 2753 3774 hsa-miR-523-5p2754 3775 hsa-miR-524-3p 2755 3776 colon cancer stem cellshsa-miR-524-5p 2756 3777 placental specific gliomas hsa-miR-525-3p 27573778 placental specific HCC hsa-miR-525-5p 2758 3779 placental specifichsa-miR-526a 2759 3780 placental specific hsa-miR-526b-3p 2760 3781placental specific hsa-miR-526b-5p 2761 3782 placental specifichsa-miR-527 2762 3783 hsa-miR-532-3p 2763 3784 ALL hsa-miR-532-5p 27643785 ALL hsa-miR-539-3p 2765 3786 hsa-miR-539-5p 2766 3787hsa-miR-541-3p 2767 3788 hsa-miR-541-5p 2768 3789 hsa-miR-542-3p 27693790 monocytes hsa-miR-542-5p 2770 3791 basal cell carcinoma,neuroblastoma hsa-miR-543 2771 3792 hsa-miR-544a 2772 3793 osteocarcomahsa-miR-544b 2773 3794 osteocarcoma hsa-miR-545-3p 2774 3795hsa-miR-545-5p 2775 3796 rectal cancer hsa-miR-548 2776 3797hsa-miR-548-3p 2777 3798 hsa-miR-548-5p 2778 3799 hsa-miR-548a 2779 3800identified in colorectal microRNAome hsa-miR-548a-3p 2780 3801identified in colorectal microRNAome hsa-miR-548a-5p 2781 3802identified in colorectal microRNAome hsa-miR-548aa 2782 3803 identifiedin cervical tumor hsa-miR-548ab 2783 3804 discovered in B- cellshsa-miR-548ac 2784 3805 discovered in B- cells hsa-miR-548ad 2785 3806discovered in B- cells hsa-miR-548ae 2786 3807 discovered in B- cellshsa-miR-548ag 2787 3808 discovered in B- cells hsa-miR-548ah-3p 27883809 discovered in B- cells hsa-miR-548ah-5p 2789 3810 discovered in B-cells hsa-miR-548ai 2790 3811 discovered in B- cells hsa-miR-548aj-3p2791 3812 discovered in B- cells hsa-miR-548aj-5p 2792 3813 discoveredin B- cells hsa-miR-548ak 2793 3814 discovered in B- cells hsa-miR-548al2794 3815 discovered in B- cells hsa-miR-548am-3p 2795 3816 discoveredin B- cells hsa-miR-548am-5p 2796 3817 discovered in B- cellshsa-miR-548an 2797 3818 discovered in B- cells hsa-miR-548ao-3p 27983819 hsa-miR-548ao-5p 2799 3820 hsa-miR-548ap-3p 2800 3821hsa-miR-548ap-5p 2801 3822 hsa-miR-548aq-3p 2802 3823 hsa-miR-548aq-5p2803 3824 hsa-miR-548ar-3p 2804 3825 hsa-miR-548ar-5p 2805 3826hsa-miR-548as-3p 2806 3827 hsa-miR-548as-5p 2807 3828 hsa-miR-548at-3p02808 3829 prostate cancer hsa-miR-548at-5p 2809 3830 prostate cancerhsa-miR-548au-3p 2810 3831 hsa-miR-548au-5p 2811 3832 hsa-miR-548av-3p2812 3833 hsa-miR-548av-5p 2813 3834 hsa-miR-548aw 2814 3835 prostatecancer hsa-miR-548ay-3p 2815 3836 discovered in abnormal skin(psoriasis) hsa-miR-548ay-5p 2816 3837 discovered in abnormal skin(psoriasis) hsa-miR-548az-3p 2817 3838 discovered in abnormal skin(psoriasis) hsa-miR-548az-5p 2818 3839 discovered in abnormal skin(psoriasis) hsa-miR-548b-3p 2819 3840 identified in colorectalmicroRNAome hsa-miR-548b-5p 2820 3841 immune cells, frontal cortexhsa-miR-548c-3p 2821 3842 identified in colorectal microRNAomehsa-miR-548c-5p 2822 3843 immune cells, frontal cortex hsa-miR-548d-3p2823 3844 identified in colorectal microRNAome hsa-miR-548d-5p 2824 3845identified in colorectal microRNAome hsa-miR-548e 2825 3846 embryonicstem cells hsa-miR-548f 2826 3847 embryonic stem cells hsa-miR-548g-3p2827 3848 embryonic stem cells hsa-miR-548g-5p 2828 3849 embryonic stemcells hsa-miR-548h-3p 2829 3850 embryonic stem cells hsa-miR-548h-5p2830 3851 embryonic stem cells hsa-miR-548i 2831 3852 embryonic stemcells, immune cells hsa-miR-548j 2832 3853 immune cells hsa-miR-548k2833 3854 embryonic stem cells hsa-miR-5481 2834 3855 embryonic stemcells hsa-miR-548m 2835 3856 embryonic stem cells hsa-miR-548n 2836 3857embryonic stem cells, immune cells hsa-miR-548o-3p 2837 3858 embryonicstem cells hsa-miR-548o-5p 2838 3859 embryonic stem cells hsa-miR-548p2839 3860 embryonic stem cells hsa-miR-548q 2840 3861 ovarian cancercells hsa-miR-548s 2841 3862 discovered in the melanoma MicroRNAomehsa-miR-548t-3p 2842 3863 discovered in the melanoma MicroRNAomehsa-miR-548t-5p 2843 3864 discovered in the melanoma MicroRNAomehsa-miR-548u 2844 3865 discovered in the melanoma MicroRNAomehsa-miR-548w 2845 3866 discovered in the melanoma MicroRNAomehsa-miR-548y 2846 3867 / hsa-miR-548z 2847 3868 discovered in cervicaltumor hsa-miR-549a 2848 3869 discovered in a colorectal MicroRNAomehsa-miR-550a-3-5p 2849 3870 Hepatocellular Carcinoma hsa-miR-550a-3p2850 3871 Hepatocellular Carcinoma hsa-miR-550a-5p 2851 3872Hepatocellular Carcinoma hsa-miR-550b-2-5p 2852 3873 discovered incervical tumor hsa-miR-550b-3p 2853 3874 discovered in cervical tumorhsa-miR-551a 2854 3875 gastric cancer hsa-miR-551b-3p 2855 3876hepatocytes hsa-miR-551b-5p 2856 3877 hepatocytes hsa-miR-552 2857 3878discovered in a colorectal MicroRNAome hsa-miR-553 2858 3879 discoveredin a colorectal MicroRNAome hsa-miR-554 2859 3880 discovered in acolorectal MicroRNAome hsa-miR-555 2860 3881 discovered in a colorectalMicroRNAome hsa-miR-556-3p 2861 3882 discovered in a colorectalMicroRNAome hsa-miR-556-5p 2862 3883 discovered in a colorectalMicroRNAome hsa-miR-557 2863 3884 liver(hepatocytes) hsa-miR-5571-3p2864 3885 discoveredd in Salivary gland hsa-miR-5571-5p 2865 3886discoveredd in Salivary gland hsa-miR-5572 2866 3887 discoveredd inSalivary gland hsa-miR-5579-3p 2867 3888 hsa-miR-5579-5p 2868 3889hsa-miR-558 2869 3890 neuroblastoma hsa-miR-5580-3p 2870 3891hsa-miR-5580-5p 2871 3892 hsa-miR-5581-3p 2872 3893 hsa-miR-5581-5p 28733894 hsa-miR-5582-3p 2874 3895 hsa-miR-5582-5p 2875 3896 hsa-miR-5583-3p2876 3897 hsa-miR-5583-5p 2877 3898 hsa-miR-5584-3p 2878 3899hsa-miR-5584-5p 2879 3900 hsa-miR-5585-3p 2880 3901 hsa-miR-5585-5p 28813902 hsa-miR-5586-3p 2882 3903 hsa-miR-5586-5p 2883 3904 hsa-miR-5587-3p2884 3905 hsa-miR-5587-5p 2885 3906 hsa-miR-5588-3p 2886 3907hsa-miR-5588-5p 2887 3908 hsa-miR-5589-3p 2888 3909 hsa-miR-5589-5p 28893910 hsa-miR-559 2890 3911 hsa-miR-5590-3p 2891 3912 hsa-miR-5590-5p2892 3913 hsa-miR-5591-3p 2893 3914 hsa-miR-5591-5p 2894 3915hsa-miR-561-3p 2895 3916 multiple myeloma hsa-miR-561-5p 2896 3917multiple myeloma hsa-miR-562 2897 3918 hsa-miR-563 2898 3919 discoveredin a colorectal MicroRNAome hsa-miR-564 2899 3920 Chronic myeloidleukemia hsa-miR-566 2900 3921 MALT lymphoma/lymphocyte hsa-miR-567 29013922 colorectal cancer hsa-miR-568 2902 3923 discovered in a colorectalMicroRNAome hsa-miR-5680 2903 3924 Associated with metastatic prostatecancer hsa-miR-5681a 2904 3925 Associated with metastatic prostatecancer hsa-miR-5681b 2905 3926 Associated with metastatic prostatecancer hsa-miR-5682 2906 3927 Associated with metastatic prostate cancerhsa-miR-5683 2907 3928 Associated with metastatic prostate cancerhsa-miR-5684 2908 3929 Associated with metastatic prostate cancerhsa-miR-5685 2909 3930 Associated with metastatic prostate cancerhsa-miR-5686 2910 3931 Associated with metastatic prostate cancerhsa-miR-5687 2911 3932 Associated with metastatic prostate cancerhsa-miR-5688 2912 3933 Associated with metastatic prostate cancerhsa-miR-5689 2913 3934 Associated with metastatic prostate cancerhsa-miR-569 2914 3935 hsa-miR-5690 2915 3936 Associated with metastaticprostate cancer hsa-miR-5691 2916 3937 Associated with metastaticprostate cancer hsa-miR-5692a 2917 3938 Associated with metastaticprostate cancer hsa-miR-5692b 2918 3939 Associated with metastaticprostate cancer hsa-miR-5692c 2919 3940 Associated with metastaticprostate cancer hsa-miR-5693 2920 3941 Associated with metastaticprostate cancer hsa-miR-5694 2921 3942 Associated with metastaticprostate cancer hsa-miR-5695 2922 3943 Associated with metastaticprostate cancer hsa-miR-5696 2923 3944 Associated with metastaticprostate cancer hsa-miR-5697 2924 3945 Associated with metastaticprostate cancer hsa-miR-5698 2925 3946 Associated with metastaticprostate cancer hsa-miR-5699 2926 3947 Associated with metastaticprostate cancer hsa-miR-5700 2927 3948 Associated with metastaticprostate cancer hsa-miR-5701 2928 3949 Associated with metastaticprostate cancer hsa-miR-5702 2929 3950 Associated with metastaticprostate cancer hsa-miR-5703 2930 3951 Associated with metastaticprostate cancer hsa-miR-570-3p 2931 3952 follicular lymphomahsa-miR-5704 2932 3953 Associated with metastatic prostate cancerhsa-miR-5705 2933 3954 Associated with metastatic prostate cancerhsa-miR-570-5p 2934 3955 follicular lymphoma hsa-miR-5706 2935 3956Associated with metastatic prostate cancer hsa-miR-5707 2936 3957Associated with metastatic prostate cancer hsa-miR-5708 2937 3958Associated with metastatic prostate cancer hsa-miR-571 2938 3959 frontalcortex hsa-miR-572 2939 3960 circulating basal cell microRNA (incarcinoma plasma) hsa-miR-573 2940 3961 discovered in the colorectalMicroRNAome hsa-miR-5739 2941 3962 endothelial cells hsa-miR-574-3p 29423963 blood (myeloid follicular cells) lymphoma hsa-miR-574-5p 2943 3964semen hsa-miR-575 2944 3965 gastric cancer hsa-miR-576-3p 2945 3966discovered in a colorectal MicroRNAome hsa-miR-576-5p 2946 3967cartilage/chondrocyte hsa-miR-577 2947 3968 discovered in a colorectalMicroRNAome hsa-miR-578 2948 3969 discovered in a colorectal MicroRNAomehsa-miR-5787 2949 3970 fibroblast hsa-miR-579 2950 3971 hsa-miR-580 29513972 breast cancer hsa-miR-581 2952 3973 liver(hepatocytes)hsa-miR-582-3p 2953 3974 cartilage/ bladder cancer chondrocytehsa-miR-582-5p 2954 3975 bladder cancer hsa-miR-583 2955 3976 rectalcancer cells hsa-miR-584-3p 2956 3977 tumor cells (follicular lymphoma,rectal cancer cells) hsa-miR-584-5p 2957 3978 tumor cells (follicularlymphoma, rectal cancer cells) hsa-miR-585 2958 3979 oral squamous cellcarcinoma hsa-miR-586 2959 3980 discovered in a colorectal MicroRNAomehsa-miR-587 2960 3981 discovered in a colorectal MicroRNAome hsa-miR-5882961 3982 discovered in a colorectal MicroRNAome hsa-miR-589-3p 29623983 mesothelial cells hsa-miR-589-5p 2963 3984 mesothelial cellshsa-miR-590-3p 2964 3985 cardiomyocytes Cell cycle progressionhsa-miR-590-5p 2965 3986 cardiomyocytes Cell cycle progressionhsa-miR-591 2966 3987 neuroblastoma hsa-miR-592 2967 3988 hepatocellularcarcinoma hsa-miR-593-3p 2968 3989 esophageal cancer hsa-miR-593-5p 29693990 esophageal cancer hsa-miR-595 2970 3991 heart failure hsa-miR-5962971 3992 ependymoma, cancers hsa-miR-597 2972 3993 discovered in acolorectal MicroRNAome hsa-miR-598 2973 3994 Blood (lymphocytes)hsa-miR-599 2974 3995 Multiple sclerosis hsa-miR-600 2975 3996discovered in a colorectal MicroRNAome hsa-miR-601 2976 3997 variouscancers (colonrectal, gastric) hsa-miR-602 2977 3998 oocyte hsa-miR-6032978 3999 hsa-miR-604 2979 4000 discovered in a colorectal MicroRNAomehsa-miR-605 2980 4001 discovered in a colorectal MicroRNAome hsa-miR-6062981 4002 discovered in a colorectal MicroRNAome hsa-miR-6068 2982 4003discovered in endothelial cells hsa-miR-6069 2983 4004 discovered inendothelial cells hsa-miR-607 2984 4005 discovered in a colorectalMicroRNAome hsa-miR-6070 2985 4006 discovered in a colorectalMicroRNAome hsa-miR-6071 2986 4007 discovered in endothelial cellshsa-miR-6072 2987 4008 discovered in endothelial cells hsa-miR-6073 29884009 discovered in endothelial cells hsa-miR-6074 2989 4010 discoveredin endothelial cells hsa-miR-6075 2990 4011 discovered in endothelialcells hsa-miR-6076 2991 4012 discovered in endothelial cellshsa-miR-6077 2992 4013 discovered in endothelial cells hsa-miR-6078 29934014 discovered in endothelial cells hsa-miR-6079 2994 4015 discoveredin endothelial cells hsa-miR-608 2995 4016 various cancers hsa-miR-60802996 4017 discovered in endothelial cells hsa-miR-6081 2997 4018discovered in endothelial cells hsa-miR-6082 2998 4019 discovered inendothelial cells hsa-miR-6083 2999 4020 discovered in endothelial cellshsa-miR-6084 3000 4021 discovered in endothelial cells hsa-miR-6085 30014022 discovered in endothelial cells hsa-miR-6086 3002 4023 embryonicstem cells hsa-miR-6087 3003 4024 embryonic stem cells hsa-miR-6088 30044025 embryonic stem cells hsa-miR-6089 3005 4026 embryonic stem cellshsa-miR-609 3006 4027 discovered in a colorectal MicroRNAomehsa-miR-6090 3007 4028 embryonic stem cells hsa-miR-610 3008 4029gastric cancer hsa-miR-611 3009 4030 Renal cell carcinoma hsa-miR-6123010 4031 AM leukemia hsa-miR-6124 3011 4032 hsa-miR-6125 3012 4033hsa-miR-6126 3013 4034 hsa-miR-6127 3014 4035 hsa-miR-6128 3015 4036hsa-miR-6129 3016 4037 hsa-miR-613 3017 4038 lipid metabollismhsa-miR-6130 3018 4039 hsa-miR-6131 3019 4040 hsa-miR-6132 3020 4041hsa-miR-6133 3021 4042 hsa-miR-6134 3022 4043 hsa-miR-614 3023 4044circulating micrRNAs (in Plasma) hsa-miR-615-3p 3024 4045 hsa-miR-615-5p3025 4046 hsa-miR-616-3p 3026 4047 prostate cancer hsa-miR-6165 30274048 Pro-apoptotic factor hsa-miR-616-5p 3028 4049 prostate cancerhsa-miR-617 3029 4050 hsa-miR-618 3030 4051 hsa-miR-619 3031 4052discovered in a colorectal MicroRNAome hsa-miR-620 3032 4053 discoveredin a colorectal MicroRNAome hsa-miR-621 3033 4054 hsa-miR-622 3034 4055hsa-miR-623 3035 4056 hsa-miR-624-3p 3036 4057 chondrocytehsa-miR-624-5p 3037 4058 chondrocyte hsa-miR-625-3p 3038 4059liver(hepatocytes), various cancers circulating (blood) hsa-miR-625-5p3039 4060 liver(hepatocytes), various cancers circulating (blood)hsa-miR-626 3040 4061 discovered in the colorectal MicroRNAomehsa-miR-627 3041 4062 colorectal cancer hsa-miR-628-3p 3042 4063neuroblastoma hsa-miR-628-5p 3043 4064 neuroblastoma hsa-miR-629-3p 30444065 B-lineage ALL, T cell lupus, RCC/kidney hsa-miR-629-5p 3045 4066B-lineage ALL, T cell lupus, RCC/kidney hsa-miR-630 3046 4067chondrocytes rectal cancer hsa-miR-631 3047 4068 discovered in thecolorectal MicroRNAom hsa-miR-632 3048 4069 myelodysplastic syndromeshsa-miR-633 3049 4070 multiple sclerosis hsa-miR-634 3050 4071cartilage/chondrocyte hsa-miR-635 3051 4072 discovered in the colorectalMicroRNAome hsa-miR-636 3052 4073 myelodysplastic syndromes hsa-miR-6373053 4074 discovered in the colorectal MicroRNAome hsa-miR-638 3054 4075Lupus nephritis, basal cell carcinoma hsa-miR-639 3055 4076 discoveredin the colorectal MicroRNAome hsa-miR-640 3056 4077 Chronic lymphocyticleukemia hsa-miR-641 3057 4078 cartilage/chondrocyte hsa-miR-642a-3p3058 4079 adipocyte hsa-miR-642a-5p 3059 4080 discovered in thecolorectal MicroRNAome hsa-miR-642b-3p 3060 4081 discovered in a cervialtumo hsa-miR-642b-5p 3061 4082 discovered in a cervial tumo hsa-miR-6433062 4083 discovered in the colorectal MicroRNAome hsa-miR-644a 30634084 hsa-miR-645 3064 4085 ovarian cancer hsa-miR-646 3065 4086hsa-miR-647 3066 4087 prostate and lung cancer hsa-miR-648 3067 4088circulating micrRNAs (in Plasma) hsa-miR-649 3068 4089 Serumhsa-miR-6499-3p 3069 4090 discovered in abnormal skin (psoriasis)hsa-miR-6499-5p 3070 4091 discovered in abnormal skin (psoriasis)hsa-miR-650 3071 4092 melanoma hsa-miR-6500-3p 3072 4093 discovered inabnormal skin (psoriasis) hsa-miR-6500-5p 3073 4094 discovered inabnormal skin (psoriasis) hsa-miR-6501-3p 3074 4095 discovered inabnormal skin (psoriasis) hsa-miR-6501-5p 3075 4096 discovered inabnormal skin (psoriasis) hsa-miR-6502-3p 3076 4097 discovered inabnormal skin (psoriasis) hsa-miR-6502-5p 3077 4098 discovered inabnormal skin (psoriasis) hsa-miR-6503-3p 3078 4099 discovered inabnormal skin (psoriasis) hsa-miR-6503-5p 3079 4100 discovered inabnormal skin (psoriasis) hsa-miR-6504-3p 3080 4101 discovered inabnormal skin (psoriasis) hsa-miR-6504-5p 3081 4102 discovered inabnormal skin (psoriasis) hsa-miR-6505-3p 3082 4103 discovered inabnormal skin (psoriasis) hsa-miR-6505-5p 3083 4104 discovered inabnormal skin (psoriasis) hsa-miR-6506-3p 3084 4105 discovered inabnormal skin (psoriasis) hsa-miR-6506-5p 3085 4106 discovered inabnormal skin (psoriasis) hsa-miR-6507-3p 3086 4107 discovered inabnormal skin (psoriasis) hsa-miR-6507-5p 3087 4108 discovered inabnormal skin (psoriasis) hsa-miR-6508-3p 3088 4109 discovered inabnormal skin (psoriasis) hsa-miR-6508-5p 3089 4110 discovered inabnormal skin (psoriasis) hsa-miR-6509-3p 3090 4111 discovered inabnormal skin (psoriasis) hsa-miR-6509-5p 3091 4112 discovered inabnormal skin (psoriasis) hsa-miR-651 3092 4113 discovered in the lungcancer colorectal MicroRNAome hsa-miR-6510-3p 3093 4114 discovered inabnormal skin (psoriasis) hsa-miR-6510-5p 3094 4115 discovered inabnormal skin (psoriasis) hsa-miR-6511a-3p 3095 4116 discovered inabnormal skin (psoriasis) and epididymis hsa-miR-6511a-5p 3096 4117discovered in abnormal skin (psoriasis) and epididymis hsa-miR-6511b-3p3097 4118 discovered in epididymis hsa-miR-6511b-5p 3098 4119 discoveredin epididymis hsa-miR-6512-3p 3099 4120 discovered in abnormal skin(psoriasis) hsa-miR-6512-5p 3100 4121 discovered in abnormal skin(psoriasis) hsa-miR-6513-3p 3101 4122 discovered in abnormal skin(psoriasis) hsa-miR-6513-5p 3102 4123 discovered in abnormal skin(psoriasis) hsa-miR-6514-3p 3103 4124 discovered in abnormal skin(psoriasis) hsa-miR-6514-5p 3104 4125 discovered in abnormal skin(psoriasis) hsa-miR-6515-3p 3105 4126 discovered in abnormal skin(psoriasis) and epididymis hsa-miR-6515-5p 3106 4127 discovered inabnormal skin (psoriasis) and epididymis hsa-miR-652-3p 3107 4128 rectalcancer cells hsa-miR-652-5p 3108 4129 rectal cancer cells hsa-miR-6533109 4130 Discovered in the colorectal MicroRNAome hsa-miR-654-3p 31104131 Discovered in the colorectal MicroRNAome hsa-miR-654-5p 3111 4132bone marrow prostate cancer hsa-miR-655 3112 4133 hsa-miR-656 3113 4134various cancers hsa-miR-657 3114 4135 oligodendrocytes diabeteshsa-miR-658 3115 4136 gastric cancer hsa-miR-659-3p 3116 4137 myoblasthsa-miR-659-5p 3117 4138 myoblast hsa-miR-660-3p 3118 4139 myoblasthsa-miR-660-5p 3119 4140 myoblast hsa-miR-661 3120 4141 breast cancerhsa-miR-662 3121 4142 endothelial progenitor cells, oocytes hsa-miR-663a3122 4143 follicular lymphoma, Lupus nephritis hsa-miR-663b 3123 4144follicular lymphoma, Lupus nephritis hsa-miR-664a-3p 3124 4145 embryonicstem component of cells SnoRNAs hsa-miR-664a-5p 3125 4146 embryonic stemcomponent of cells SnoRNAs hsa-miR-664b-3p 3126 4147 embryonic stemcomponent of cells SnoRNAs hsa-miR-664b-5p 3127 4148 embryonic stemcomponent of cells SnoRNAs hsa-miR-665 3128 4149 breast cancerhsa-miR-668 3129 4150 keratinocytes senescence hsa-miR-670 3130 4151hsa-miR-671-3p 3131 4152 hsa-miR-6715a-3p 3132 4153 discovered inepididymis hsa-miR-6715b-3p 3133 4154 discovered in epididymishsa-miR-6715b-5p 3134 4155 discovered in epididymis hsa-miR-671-5p 31354156 rectal cancer, prolactinomas hsa-miR-6716-3p 3136 4157 discoveredin epididymis hsa-miR-6716-5p 3137 4158 discovered in epididymishsa-miR-6717-5p 3138 4159 discovered in epididymis hsa-miR-6718-5p 31394160 discovered in epididymis hsa-miR-6719-3p 3140 4161 discovered inepididymis hsa-miR-6720-3p 3141 4162 discovered in epididymishsa-miR-6721-5p 3142 4163 discovered in epididymis hsa-miR-6722-3p 31434164 discovered in epididymis hsa-miR-6722-5p 3144 4165 discovered inepididymis hsa-miR-6723-5p 3145 4166 discovered in epididymishsa-miR-6724-5p 3146 4167 discovered in epididymis hsa-miR-675-3p 31474168 adrenocortical tumor hsa-miR-675-5p 3148 4169 adrenocortical tumorhsa-miR-676-3p 3149 4170 discovered in female reproductuve tracthsa-miR-676-5p 3150 4171 discovered in female reproductuve tracthsa-miR-708-3p 3151 4172 Various cancers (lung, bladder, pancreatic,ALL) hsa-miR-708-5p 3152 4173 Various cancers (lung, bladder,pancreatic, ALL) hsa-miR-711 3153 4174 cutaneous T-cell lymphomashsa-miR-7-1-3p 3154 4175 Glioblast, brain, prancreas hsa-miR-718 31554176 blood hsa-miR-7-2-3p 3156 4177 brain, pancreas hsa-miR-744-3p 31574178 heart hsa-miR-744-5p 3158 4179 embryonic stem cells, hearthsa-miR-758-3p 3159 4180 cholesterol regulation and brain hsa-miR-758-5p3160 4181 cholesterol regulation and brain hsa-miR-759 3161 4182hsa-miR-7-5p 3162 4183 brain hsa-miR-760 3163 4184 colonrectal andbreast cancer hsa-miR-761 3164 4185 hsa-miR-762 3165 4186 cornealepithelial cells hsa-miR-764 3166 4187 osteoblast hsa-miR-765 3167 4188rectal cancer hsa-miR-766-3p 3168 4189 embryonic stem cellshsa-miR-766-5p 3169 4190 embryonic stem cells hsa-miR-767-3p 3170 4191hsa-miR-767-5p 3171 4192 hsa-miR-769-3p 3172 4193 hsa-miR-769-5p 31734194 hsa-miR-770-5p 3174 4195 hsa-miR-802 3175 4196 brain, epithelialdown symdrome cells, hepatocytes hsa-miR-873-3p 3176 4197 hsa-miR-873-5p3177 4198 hsa-miR-874 3178 4199 cervical cancer, lung cancer, carcinomahsa-miR-875-3p 3179 4200 hsa-miR-875-5p 3180 4201 hsa-miR-876-3p 31814202 hsa-miR-876-5p 3182 4203 hsa-miR-877-3p 3183 4204 hsa-miR-877-5p3184 4205 hsa-miR-885-3p 3185 4206 embryonic stem cells hsa-miR-885-5p3186 4207 embryonic stem cells hsa-miR-887 3187 4208 hsa-miR-888-3p 31884209 hsa-miR-888-5p 3189 4210 hsa-miR-889 3190 4211 hsa-miR-890 31914212 epididymis hsa-miR-891a 3192 4213 epididymis osteosarcomahsa-miR-891b 3193 4214 epididymis hsa-miR-892a 3194 4215 epididymishsa-miR-892b 3195 4216 epididymis hsa-miR-892c-3p 3196 4217 discoveredin epididymis hsa-miR-892c-5p 3197 4218 discovered in epididymishsa-miR-920 3198 4219 human testis hsa-miR-921 3199 4220 human testismuscle invasive bladder cancer hsa-miR-922 3200 4221 human testis,multiple sclerosis, neuronal tissues Alcoholic liver disease hsa-miR-9243201 4222 human testis hsa-miR-92a-1-5p 3202 4223 endothelial cellshsa-miR-92a-2-5p 3203 4224 endothelial cells hsa-miR-92a-3p 3204 4225endothelial cells, CNS hsa-miR-92b-3p 3205 4226 endothelial cells, hearthsa-miR-92b-5p 3206 4227 endothelial cells, heart hsa-miR-933 3207 4228discovered in cervical cancer hsa-miR-93-3p 3208 4229 embryonic stembasal cell cells carcinoma hsa-miR-934 3209 4230 discovered in cervicalcancer hsa-miR-935 3210 4231 blood monoclear energy cells metabolism/obesity, medullablastoma/neural stem cells hsa-miR-93-5p 3211 4232embryonic stem cells hsa-miR-936 3212 4233 skin hsa-miR-937-3p 3213 4234cervical cancer hsa-miR-937-5p 3214 4235 cervical cancer hsa-miR-9383215 4236 Various cancer cells hsa-miR-939-3p 3216 4237 hepatocyteshsa-miR-939-5p 3217 4238 hepatocytes hsa-miR-9-3p 3218 4239 brainCancers and brain diseases hsa-miR-940 3219 4240 identified in Cervicalcancer hsa-miR-941 3220 4241 Embryonic stem cells hsa-miR-942 3221 4242lung cancer hsa-miR-943 3222 4243 identified in Cervical cancerhsa-miR-944 3223 4244 various cancers (cervical, pancreatic,colonrectal) hsa-miR-95 3224 4245 various cancers (pancreatic,glioblastoma, colorectal etc) hsa-miR-9-5p 3225 4246 brain Cancers andbrain disease hsa-miR-96-3p 3226 4247 stem cells various cancers(prostate, lymphoma, HCC, etc) and inflammation hsa-miR-96-5p 3227 4248stem cells various cancers (prostate, lymphoma, HCC, etc) andinflammation hsa-miR-98-3p 3228 4249 various cancer apoptosis cellshsa-miR-98-5p 3229 4250 various cancer apoptosis cells hsa-miR-99a-3p3230 4251 hemapoietic cells hsa-miR-99a-5p 3231 4252 hemapoietic cellshsa-miR-99b-3p 3232 4253 hemapoietic cells, embryonic stem cellshsa-miR-99b-5p 3233 4254 hemapoietic cells, embryonic stem cells

MicroRNAs that are enriched in specific types of immune cells are listedin Table 13. Furthermore, novel miroRNAs are discovered in the immunecells in the art through micro-array hybridization and microtomeanalysis (Jima D D et al, Blood, 2010, 116:e118-e127; Vaz C et al., BMCGenomics, 2010, 11,288, the content of each of which is incorporatedherein by reference in its entirety). In Table 13, “HCC” representshepatocellular carcinoma, “ALL” stands for acute lymphoblastsic leukemiaand “CLL” stands for chrominc lymphocytic leukemia.

TABLE 13 microRNAs in immune cells mir BS SEQ SEQ tissues/cells withassociated biological microRNA ID ID MicroRNAs diseasesfunctions/targets hsa-let-7a-2-3p 171 1192 embryonic stem inflammatory,tumor suppressor, cells, lung, various cancers target to c-myc myeloidcells (lung, cervical, breast, pancreatic, etc) hsa-let-7a-3p 172 1193embryonic stem inflammatory, tumor suppressor, cell, lung, variouscancers target to c-myc myeloid cells (lung, cervical, breast,pancreatic, etc) hsa-let-7a-5p 173 1194 embryonic stem inflammatory,tumor suppressor, cells, lung, various cancers target to c-myc myeloidcells (lung, cervical, breast, pancreatic, etc) hsa-let-7c 176 1197dendritic cells various cacners tumor suppressor (cervical, apoptosis(target pancreatic, to BCL-xl) lung, esopphageal, etc) hsa-let-7e-3p 1791200 immune cells various cancer tumor suppressor cells, autoimmunityTLR signal pathway in endotoxin tolerance hsa-let-7e-5p 180 1201 immunecells associated with tumor suppressor various cancer cellshsa-let-7f-1-3p 181 1202 immune cells (T associated with tumorsuppressor cells) various cancer cells hsa-let-7f-2-3p 182 1203 immunecells (T associated with tumor suppressor cells) various cancer cellshsa-let-7f-5p 183 1204 immune cells (T associated with tumor suppressorcells) various cancer cells hsa-let-7g-3p 184 1205 hematopoietic variouscancer tumor suppressor cells, adipose, cells (lung, (target to NFkB,smooth muscle breast, etc) LOX1 cells hsa-let-7g-5p 185 1206hematopoietic various cancer tumor suppressor cells, adipose, cells(lung, (target to NFkB, smooth muscle breast, etc) LOX1 cellshsa-let-7i-3p 186 1207 immune cells chronic tumor suppressor lymphocyteleukimia hsa-let-7i-5p 187 1208 immune cells chronic tumor suppressorlymphocyte leukimia hsa-miR-10a- 203 1224 hematopoeitic acute myeoidoncogene, cell 3p cells leukemia growth hsa-miR-10a- 204 1225hematopoietic acute myeloid oncogene, cell 5p cells leukemia growthhsa-miR-1184 214 1235 Hematopoietic downregulated in predited in thecells oral leukoplakia intron 22 of F8 (OLK) gene hsa-miR-125b- 279 1300hematopoietic various cancer oncogene, cell 1-3p cells (ALL, prostate,differentiation (monocytes), HCC, etc); TLR brain (neuron) signalpathway in endotoxin tolerance hsa-miR-125b- 280 1301 hematopoieticvarious cancer oncogene cell 2-3p cells (ALL, prostate, differentiation(monocytes), HCC etc); TLR brain (neuron) signal pathway in endotoxintolerance hsa-miR-125b- 281 1302 hematopoietic various cancer oncogenecell 5p cells, brain (Cutaneous T cell differentiation (neuron)lymphomas, prostate, HCC, etc); TLR signal pathway in endotoxintolerance hsa-miR-1279 315 1336 monocytes hsa-miR-130a- 353 1374 lung,monocytes, various cancers pro-angiogenic 3p vascular (basal cellendothelial cells carcinoma, HCC, ovarian, etc), drug resistancehsa-miR-130a- 354 1375 lung, monocytes, various cancers pro-angiogenic5p vasscular (basal cell endothelial cells carcinoma, HCC, ovarian,etc), drug resistance hsa-miR-132- 360 1381 brain (neuron), 3p immunecells hsa-miR-132- 362 1383 brain (neuron), 5p immune cells hsa-miR-142-383 1404 meyloid cells, tumor suppressor, 3p hematopoiesis, immuneresponse APC cells hsa-miR-142- 384 1405 meyloid cells, immune response5p hematopoiesis, APC cells hsa-miR-143- 386 1407 vascular smoothincreased in 5p muscle, T-cells serum after virus infectionhsa-miR-146a- 393 1414 immune cells, associated with 3p hematopoiesis,cartilage, CLL, TLR signal pathway in endotoxin tolerance hsa-miR-146a-394 1415 immune cells, associated with 5p hematopoiesis, CLL, TLR signalcartilage, pathway in endotoxin tolerance hsa-miR-146b- 395 1416 immunecells cancers (thyroid immune response 3p carcimona) hsa-miR-146b- 3961417 embryoid body thyroid cancer, tumor invation, 5p cells associatedwith migration CLL hsa-miR-147a 399 1420 Macrophage inflammatoryresponse hsa-miR-147b 400 1421 Macrophage inflammatory responsehsa-miR-148a- 401 1422 hematopoietic associated with 3p cells CLL,T-lineage ALL hsa-miR-148a- 402 1423 hematopoietic associated with 5pcells CLL, T-lineage ALL hsa-miR-150- 407 1428 hematopoitic circulating3p cells (lymphoid) plasma (acute myeloid leukemia) hsa-miR-150- 4081429 hematopoitic circulating 5p cells (lymphoid) plasma (acute myeloidleukemia) hsa-miR-151b 411 1432 immune cells (B- cells) hsa-miR-155- 4191440 T/B cells, associated with 3p monocytes, breast CLL, TLR signalpathway in endotoxin tolerance; upregulated in B cell lymphoma (CLL) andother cancers (breast, lung, ovarian, cervical, colorectal, prostate)hsa-miR-155- 420 1441 T/B cells, associated with 5p monocytes, breastCLL, TLR signal pathway in endotoxin tolerance, upregulated in B celllymphoma (CLL) and other cancers (breast, lung, ovarian, cervical,colorectal, prostate) hsa-miR-15a- 422 1443 blood, chronic 3plymphocyte, lymphocytic hematopoietic leukemia tissues (spleen)hsa-miR-15a- 423 1444 blood, chronic 5p lymphocyte, lymphocytichematopoietic leukemia tissues (spleen) hsa-miR-15b- 424 1445 blood,cell cycle, 3p lymphocyte, proliferation hematopoietic tissues (spleen)hsa-miR-15b- 425 1446 blood, cell cycle, 5p lymphocyte, proliferationhematopoietic tissues (spleen) hsa-miR-16-1- 426 1447 embryonic stemchronic 3p cells, blood, lymphocytic hematopoietic leukemia tissues(spleen) hsa-miR-16-2- 427 1448 blood, 3p lymphocyte, hematopoietictissues (spleen) hsa-miR-16-5p 428 1449 blood, lymphocyte, hematopoietictissues hsa-miR-181a- 432 1453 glioblast, 3p myeloid cells, Embryonicstem cells hsa-miR-181a- 433 1454 glioblast, 5p myeloid cells, Embryonicstem cells hsa-miR-182- 439 1460 immune cells colonrectal immuneresponse 3p cancer, autoimmne hsa-miR-182- 441 1462 lung, immuneautoimmune immune response 5p cells hsa-miR-197- 490 1511 blood(myeloid), various cancers 3p other tissues (thyroid tumor, leukemia,etc) hsa-miR-197- 491 1512 blood (myeloid), various cancers 5p othertissues (thyroid tumor, leukemia, etc) hsa-miR-21-3p 542 1563 glioblast,Blood autoimmune, (meyloid cells), heart diseases, liver, vascularcancers endothelial cells hsa-miR-214- 543 1564 immune cells, variouacancers immune response 3p pancreas (melanoma, pancreatic, ovarian)hsa-miR-214- 544 1565 immune cells, varioua cancers immune response 5ppancreas (melanoma, pancreatic, ovarian) hsa-miR-21-5p 546 1567 blood(myeloid autoimmune, cells), liver, heart diseases, endothelial cellscancers hsa-miR-221- 557 1578 endothelial cells, breastangiogenesis/vasculogenesis 3p immune cells cancer, upregulated inthyroid cell transformation induced by HMGA1, TLR signal pathway inendotoxin tolerance, upregulated in T cell ALL hsa-miR-221- 558 1579endothelial cells, breast angiogenesis/vasculogenesis 5p immune cellscancer, upregulated in thyroid cell transformation induced by HMGA1, TLRsignal pathway in endotoxin tolerance, upregulated in T cell ALLhsa-miR-223- 561 1582 meyloid cells associated with 3p CLL hsa-miR-223-562 1583 meyloid cells associated with 5p CLL hsa-miR-23b- 576 1597blood, myeloid cancers (renal 3p cells cancer, glioblastoma, prostate,etc) and autoimmune hsa-miR-23b- 577 1598 blood, myeloid cancers(glioblastoma, 5p cells prostate, etc) and autoimmune hsa-miR-24-1- 5791600 lung, myeloid 5p cells hsa-miR-24-2- 580 1601 lung, myeloid 5pcells hsa-miR-24-3p 581 1602 lung, myeloid cells hsa-miR-26a-1- 590 1611embryonic stem chronic cell cycle and 3p cells, blood (T lymphocytedifferentiation cells) leukemia and other cancers hsa-miR-26a-2- 5911612 blood (Tcells), chronic cell cycle and 3p other tissues lymphocytedifferentiation leukemia and other cancers hsa-miR-26a- 592 1613 blood(Tcells), chronic cell cycle and 5p other tissues lymphocytedifferentiation leukemia and other cancers hsa-miR-26b- 593 1614hematopoietic 3p cells hsa-miR-26b- 594 1615 hematopoietic 5p cellshsa-miR-27a- 595 1616 myeloid cells various cancer 3p cells hsa-miR-27a-596 1617 myeloid cells various cancer 5p cells hsa-miR-27b- 597 1618myeloid cells, various cancer pro-angiogenic 3p vascular cellsendothelial cells hsa-miR-28-3p 599 1620 blood (immune B/T cell cells)lymphoma hsa-miR-28-5p 600 1621 blood (immune B/T cell cells) lymphomahsa-miR-2909 602 1623 T-Lymphocytes hsa-miR-29a- 611 1632 immuno system,various cancers, tumor 3p colonrectun neurodegenative suppression,disease immune modulation (mir- 29 family) hsa-miR-29a- 612 1633 immunosystem, various cancers, adaptive 5p colonrectun neurodegenativeimmunity disease hsa-miR-29b- 613 1634 immuno system associated withadaptive 1-5p CLL, other immunity cancers, neurodegenative diseasehsa-miR-29b- 614 1635 immuno system associated with adaptive 2-5p CLL,other immunity cancers, hsa-miR-29b- 615 1636 immuno system associatedwith adaptive 3p CLL, other immunity cancers hsa-miR-29c- 616 1637immuno system associated with adaptive 3p CLL, other immunity cancershsa-miR-29c- 617 1638 immuno system associated with adaptive 5p CLL,other immunity cancers hsa-miR-30e- 647 1668 myeloid cells, 3p gliacells hsa-miR-30e- 648 1669 myeloid cells, 5p glia cells hsa-miR-331-793 1814 lymphocytes 5p hsa-miR-339- 800 1821 immune cells 3phsa-miR-339- 801 1822 immune cells 5p hsa-miR-345- 810 1831hematopoietic increased in 3p cells follicular lymphoma(53), othercancers hsa-miR-345- 811 1832 hematopoietic increased in 5p cellsfollicular lymphoma(53) hsa-miR-346 812 1833 immume cells cancer andautoimmune hsa-miR-34a- 813 1834 breast, myeloid gastric cancer, tumorsuppressor, 3p cells, ciliated CLL, other p53 inducible epithelial cellshsa-miR-34a- 814 1835 breast, myeloid gastric cancer, tumor suppressor,5p cells, ciliated CLL, other p53 inducible epithelial cellshsa-miR-363- 856 1877 kidney stem cell, 3p blood cells hsa-miR-363- 8571878 kidney stem cell, 5p blood cells hsa-miR-372 940 1961 hematopoieticcells, lung, placental (blood) hsa-miR-377- 957 1978 hematopoietic 3pcells hsa-miR-377- 958 1979 hematopoietic 5p cells hsa-miR-493- 26103631 myeloid cells, 3p pancreas (islet) hsa-miR-493- 2611 3632 myeloidcells, 5p pancreas (islet) hsa-miR-542- 2769 3790 monocytes targets to3p survivin, introduce growth arrest hsa-miR-548b- 2820 3841 immunecells 5p frontal cortex hsa-miR-548c- 2822 3843 immune cells 5p frontalcortex hsa-miR-548i 2831 3852 embryonic stem cells (41), immune cellshsa-miR-548j 2832 3853 immune cells hsa-miR-548n 2836 3857 embryonicstem cells, immune cells hsa-miR-574- 2942 3963 blood (myeloid increasedin 3p cells) follicular lymphoma (53) hsa-miR-598 2973 3994 in bloodlymphocytes (PBL) hsa-miR-935 3210 4231 identified in associated withhuman cervical energy cancer metabolism/obesity, blood medullablastoma/mononuclear neural stem cells cells hsa-miR-99a- 3230 4251 hemapoieticcells 3p hsa-miR-99a- 3231 4252 hemapoietic 5p cells, plasma (exosome)hsa-miR-99b- 3232 4253 hemapoietic 3p cells, Embryonic stem cells,hsa-miR-99b- 3233 4254 hemapoietic 5p cells, Embryonic stem cells,plasma(exosome)III. Modifications

Herein, in a nucleotide, nucleoside polynucleotide (such as the nucleicacids of the invention, e.g., modified RNA, modified nucleic acidmolecule, modified RNAs, nucleic acid and modified nucleic acids), theterms “modification” or, as appropriate, “modified” refer tomodification with respect to A, G, U or C ribonucleotides. Generally,herein, these terms are not intended to refer to the ribonucleotidemodifications in naturally occurring 5′-terminal mRNA cap moieties. In apolypeptide, the term “modification” refers to a modification ascompared to the canonical set of 20 amino acids.

The modifications may be various distinct modifications. In someembodiments, where the nucleic acids or modified RNA, the coding region,the flanking regions and/or the terminal regions may contain one, two,or more (optionally different) nucleoside or nucleotide modifications.In some embodiments, a modified nucleic acids or modified RNA introducedto a cell may exhibit reduced degradation in the cell, as compared to anunmodified nucleic acid or modified RNA.

The polynucleotide, primary construct, nucleic acids or modified RNA caninclude any useful modification, such as to the sugar, the nucleobase,or the internucleoside linkage (e.g. to a linking phosphate/to aphosphodiester linkage/to the phosphodiester backbone). One or moreatoms of a pyrimidine nucleobase may be replaced or substituted withoptionally substituted amino, optionally substituted thiol, optionallysubstituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro orfluoro). In certain embodiments, modifications (e.g., one or moremodifications) are present in each of the sugar and the internucleosidelinkage. Modifications according to the present invention may bemodifications of ribonucleic acids (RNAs) to deoxyribonucleic acids(DNAs), e.g., the substitution of the 2′OH of the ribofuranysyl ring to2′H, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptidenucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof).Additional modifications are described herein.

As described herein, the polynucleotides, primary construct, nucleicacids or modified RNA of the invention do not substantially induce aninnate immune response of a cell into which the polynucleotides, primaryconstructs, nucleic acids or modified RNA (e.g., mRNA) is introduced.Features of an induced innate immune response include 1) increasedexpression of pro-inflammatory cytokines, 2) activation of intracellularPRRs (RIG-I, MDA5, etc, and/or 3) termination or reduction in proteintranslation.

In certain embodiments, it may desirable for a modified nucleic acidmolecule introduced into the cell to be degraded intracellulary. Forexample, degradation of a modified nucleic acid molecule may bepreferable if precise timing of protein production is desired. Thus, insome embodiments, the invention provides a modified nucleic acidmolecule containing a degradation domain, which is capable of beingacted on in a directed manner within a cell. In another aspect, thepresent disclosure provides polynucleotides, primary constructs, nucleicacids or modified RNA comprising a nucleoside or nucleotide that candisrupt the binding of a major groove interacting, e.g. binding, partnerwith the polynucleotides, primary constructs, nucleic acids or modifiedRNA (e.g., where the modified nucleotide has decreased binding affinityto major groove interacting partner, as compared to an unmodifiednucleotide).

The polynucleotides, primary constructs, nucleic acids or modified RNAcan optionally include other agents (e.g., RNAi-inducing agents, RNAiagents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalyticDNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors,etc.). In some embodiments, the polynucleotides, primary constructs,nucleic acids or modified RNA may include one or more messenger RNAs(mRNAs) having one or more modified nucleoside or nucleotides (i.e.,modified mRNA molecules). Details for these nucleic acids or modifiedRNA follow.

Modified mRNA Molecules

The polynucleotides, primary constructs, nucleic acids or modified RNAof the invention includes a first region of linked nucleosides encodinga polypeptide of interest, a first flanking region located at the 5′terminus of the first region, and a second flanking region located atthe 3′ terminus of the first region. The first region of linkednucleosides may be a translatable region.

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having any base, sugar,backbone, building block or other structure or formula, including butnot limited to those of Formulas I through IX or any substructuresthereof as described in International Application PCT/US12/58519 filedOct. 3, 2012, the contents of which are incorporated herein by referencein their entirety. Such structures include modifications to the sugar,nucleobase, internucleoside linkage, or combinations thereof.

Combinations of chemical modifications include those taught in includingbut not limited to those described in International ApplicationPCT/US12/58519 filed Oct. 3, 2012, the contents of which areincorporated herein by reference in their entirety.

The synthesis of polynucleotides, primary constructs or mmRNA of thepresent invention may be according to the methods described inInternational Application PCT/US12/58519 filed Oct. 3, 2012, thecontents of which are incorporated herein by reference in theirentirety.

In some embodiments, the nucleobase selected from the group consistingof cytosine, guanine, adenine, and uracil.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine (s²U), 4-thio-uridine(s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine(ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or5-bromo-uridine), 3-methyluridine (m³U), 5-methoxy-uridine (mo⁵U),uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyacetic acid methyl ester(mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U), 1-carboxymethyl-pseudouridine,5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridinemethyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine(τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm⁵s²U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U,i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-β-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formylcytidine (f⁵C),N4-methylcytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine (e.g.,5-iodo-cytidine), 5-hydroxymethylcytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-aminopurine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine (m′A),2-methyl-adenine (m²A), N6-methyladenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms²m⁶A), N6-isopentenyladenosine(i⁶A), 2-methylthio-N6-isopentenyladenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyladenosine (g⁶A), N6-threonylcarbamoyladenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonyl carbamoyladenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyladenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-β-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanineExemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m^(i)I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o₂yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (O),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQo),7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methylguanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methylguanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-(m^(2,2,7)G), guanosine(m^(2,7)G), N2,N2,7-dimethyl-guanosine 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m′Im), 2′-β-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, 06-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

Phosphorothioate DNA and RNA have increased nuclease resistance andsubsequently a longer half-life in a cellular environment.Phosphorothioate linked nucleic acids are expected to also reduce theinnate immune response through weaker binding/activation of cellularinnate immune molecules.

The nucleobase of the nucleotide can be independently selected from apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can each be independently selected from adenine, cytosine,guanine, uracil, or hypoxanthine. In another embodiment, the nucleobasecan also include, for example, naturally-occurring and syntheticderivatives of a base, including pyrazolo[3,4-d]pyrimidines,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

Modifications on the Internucleoside Linkage

The modified nucleotides, which may be incorporated into a nucleic acidor modified RNA molecule, can be modified on the internucleoside linkage(e.g., phosphate backbone). Herein, in the context of thepolynucleotides, primary constructs, nucleic acids or modified RNAbackbone, the phrases “phosphate” and “phosphodiester” are usedinterchangeably. Backbone phosphate groups can be modified by replacingone or more of the oxygen atoms with a different substituent. Further,the modified nucleosides and nucleotides can include the wholesalereplacement of an unmodified phosphate moiety with anotherinternucleoside linkage as described herein. Examples of modifiedphosphate groups include, but are not limited to, phosphorothioate,phosphoroselenates, boranophosphates, boranophosphate esters, hydrogenphosphonates, phosphoramidates, phosphorodiamidates, alkyl or arylphosphonates, and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulfur. The phosphate linker can also bemodified by the replacement of a linking oxygen with nitrogen (bridgedphosphoramidates), sulfur (bridged phosphorothioates), and carbon(bridged methylene-phosphonates).

The α-thio substituted phosphate moiety is provided to confer stabilityto RNA and DNA polymers through the unnatural phosphorothioate backbonelinkages. Phosphorothioate DNA and RNA have increased nucleaseresistance and subsequently a longer half-life in a cellularenvironment. While not wishing to be bound by theory, phosphorothioatelinked polynucleotides, primary constructs, nucleic acids or modifiedRNA molecules are expected to also reduce the innate immune responsethrough weaker binding/activation of cellular innate immune molecules.

In specific embodiments, a modified nucleoside includes analpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine,5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine),5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or5′-O-(1-thiophosphate)-pseudouridine).

Other internucleoside linkages that may be employed according to thepresent invention, including internucleoside linkages which do notcontain a phosphorous atom, are described herein below.

Combinations of Modified Sugars, Nucleobases, and InternucleosideLinkages

The nucleic acids or modified RNA of the invention can include acombination of modifications to the sugar, the nucleobase, and/or theinternucleoside linkage. These combinations can include any one or moremodifications described herein. For examples, any of the nucleotidesdescribed herein in Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IVl), and (IXa)-(IXr) can be combined with any of the nucleobasesdescribed herein (e.g., in Formulas (b1)-(b43) or any other describedherein).

Synthesis of Nucleic Acids or Modified RNA Molecules (Modified RNAs)

Nucleic acids for use in accordance with the invention may be preparedaccording to any useful technique as described herein or any availabletechnique including, but not limited to chemical synthesis, enzymaticsynthesis, which is generally termed in vitro transcription, enzymaticor chemical cleavage of a longer precursor, etc. Methods of synthesizingRNAs are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.:IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in Molecular Biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporatedherein by reference).

The modified nucleosides and nucleotides used in the synthesis ofmodified RNAs disclosed herein can be prepared from readily availablestarting materials using the following general methods and procedures.It is understood that where typical or preferred process conditions(i.e., reaction temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are given; other process conditions can also be usedunless otherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry(e.g., UV-visible), or mass spectrometry, or by chromatography such ashigh performance liquid chromatography (HPLC) or thin layerchromatography.

Preparation of modified nucleosides and nucleotides used in themanufacture or synthesis of modified RNAs of the present invention caninvolve the protection and deprotection of various chemical groups. Theneed for protection and deprotection, and the selection of appropriateprotecting groups can be readily determined by one skilled in the art.

The chemistry of protecting groups can be found, for example, in Greene,et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons,1991, which is incorporated herein by reference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents, which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of modified nucleosides and nucleotidescan be carried out by any of numerous methods known in the art. Anexample method includes fractional recrystallization using a “chiralresolving acid” which is an optically active, salt-forming organic acid.Suitable resolving agents for fractional recrystallization methods are,for example, optically active acids, such as the D and L forms oftartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelicacid, malic acid, lactic acid or the various optically activecamphorsulfonic acids. Resolution of racemic mixtures can also becarried out by elution on a column packed with an optically activeresolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elutionsolvent composition can be determined by one skilled in the art.

Modified nucleosides and nucleotides (e.g., building block molecules)can be prepared according to the synthetic methods described in Ogata etal., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568(1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each ofwhich are incorporated by reference in their entirety.

Modified nucleosides and nucleotides (e.g., building block molecules)can be prepared according to the synthetic methods described in Ogata etal., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568(1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each ofwhich are incorporated by reference in their entirety.

The modified nucleic acids of the invention may or may not be uniformlymodified along the entire length of the molecule. Different nucleotidemodifications and/or backbone structures may exist at various positionsin the nucleic acid. One of ordinary skill in the art will appreciatethat the nucleotide analogs or other modification(s) may be located atany position(s) of a nucleic acid such that the function of the nucleicacid is not substantially decreased. A modification may also be a 5′ or3′ terminal modification. The nucleic acids may contain at a minimum oneand at maximum 100% modified nucleotides, or any intervening percentage,such as at least 50% modified nucleotides, at least 80% modifiednucleotides, or at least 90% modified nucleotides. For example, one ormore or all types of nucleotide (e.g., purine or pyrimidine, or any oneor more or all of A, G, U, C) may or may not be uniformly modified in anucleic acids or modified RNA of the invention, or in a givenpredetermined sequence region thereof. In some embodiments, allnucleotides X in a nucleic acids or modified RNA of the invention (or ina given sequence region thereof) are modified, wherein X may any one ofnucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C,G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

Different sugar modifications, nucleotide modifications, and/orinternucleoside linkages (e.g., backbone structures) may exist atvarious positions in the nucleic acids or modified RNA. One of ordinaryskill in the art will appreciate that the nucleotide analogs or othermodification(s) may be located at any position(s) of a nucleic acid ormodified RNA such that the function of the nucleic acids or modified RNAis not substantially decreased. A modification may also be a 5′ or 3′terminal modification. The nucleic acids or modified RNA may containfrom about 1% to about 100% modified nucleotides (either in relation tooverall nucleotide content, or in relation to one or more types ofnucleotide, i.e. any one or more of A, G, U or C) or any interveningpercentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%,from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20%to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100%).

In some embodiments, the nucleic acids or modified RNA includes amodified pyrimidine (e.g., a modified uracil/uridine/U or modifiedcytosine/cytidine/C). In some embodiments, the uracil or uridine(generally: U) in the nucleic acids or modified RNA molecule may bereplaced with from about 1% to about 100% of a modified uracil ormodified uridine (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%,from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1%to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%,from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%,from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50%to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100% of a modified uracil or modified uridine). The modified uracilor uridine can be replaced by a compound having a single uniquestructure or by a plurality of compounds having different structures(e.g., 2, 3, 4 or more unique structures, as described herein). In someembodiments, the cytosine or cytidine (generally: C) in the nucleic acidor modified RNA molecule may be replaced with from about 1% to about100% of a modified cytosine or modified cytidine (e.g., from 1% to 20%,from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1%to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%,from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%,from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50%to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to95%, from 90% to 100%, and from 95% to 100% of a modified cytosine ormodified cytidine). The modified cytosine or cytidine can be replaced bya compound having a single unique structure or by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more uniquestructures, as described herein).

Other components of the nucleic acid are optional, and are beneficial insome embodiments. For example, a 5′ untranslated region (UTR) and/or a3′UTR are provided, wherein either or both may independently contain oneor more different nucleotide modifications. In such embodiments,nucleotide modifications may also be present in the translatable region.Also provided are nucleic acids containing a Kozak sequence which mayinclude an IRES sequence or not include an IRES sequence (See e.g., thepolynucleotides described in Table 30 in Example 31).

Additionally, provided are nucleic acids containing one or more intronicnucleotide sequences capable of being excised from the nucleic acid.

Combinations of Nucleotides

Further examples of modified nucleotides and modified nucleotidecombinations are provided below in Table 14. These combinations ofmodified nucleotides can be used to form the nucleic acids or modifiedRNA of the invention. Unless otherwise noted, the modified nucleotidesmay be completely substituted for the natural nucleotides of the nucleicacids or modified RNA of the invention. As a non-limiting example, thenatural nucleotide uridine may be substituted with a modified nucleosidedescribed herein. In another non-limiting example, the naturalnucleotide uridine may be partially substituted (e.g., about 0.1%, 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 99.9%) with at least one of the modifiednucleoside disclosed herein.

TABLE 14 Chemical Modifications Modified Nucleotide Modified NucleotideCombination 6-aza-cytidine α-thio-cytidine/5-iodo-uridine2-thio-cytidine α-thio-cytidine/N1-methyl-pseudo-uridine α-thio-cytidineα-thio-cytidine/α-thio-uridine Pseudo-iso-cytidineα-thio-cytidine/5-methyl-uridine 5-aminoallyl-uridineα-thio-cytidine/pseudo-uridine 5-iodo-uridinePseudo-iso-cytidine/5-iodo-uridine N1-methyl-pseudouridinePseudo-iso-cytidine/N1-methyl-pseudo-uridine 5,6-dihydrouridinePseudo-iso-cytidine/α-thio-uridine α-thio-uridinePseudo-iso-cytidine/5-methyl-uridine 4-thio-uridinePseudo-iso-cytidine/Pseudo-uridine 6-aza-uridine 5-hydroxy-uridinePyrrolo-cytidine/5-iodo-uridine Deoxy-thymidinePyrrolo-cytidine/N1-methyl-pseudo-uridine Pseudo-uridinePyrrolo-cytidine/α-thio-uridine InosinePyrrolo-cytidine/5-methyl-uridine α-thio-guanosinePyrrolo-cytidine/Pseudo-uridine 8-oxo-guanosine5-methyl-cytidine/5-iodo-uridine O6-methyl-guanosine5-methyl-cytidine/N1-methyl-pseudo-uridine 7-deaza-guanosine5-methyl-cytidine/α-thio-uridine No modification5-methyl-cytidine/5-methyl-uridine N1-methyl-adenosine5-methyl-cytidine/Pseudo-uridine 2-amino-6-Chloro-purineN6-methyl-2-amino-purine about 25% of cytosines are Pseudo-iso-cytidine6-Chloro-purine about 25% of uridines are N1-methyl-pseudo-uridineN6-methyl-adenosine 25% N1-Methyl-pseudo-uridine/75%-pseudo-uridineα-thio-adenosine 8-azido-adenosine 7-deaza-adenosine about 50% of thecytosines are pyrrolo-cytidine Pyrrolo-cytidine5-methyl-cytidine/5-iodo-uridine 5-methyl-cytidine5-methyl-cytidine/N1-methyl-pseudouridine N4-acetyl-cytidine5-methyl-cytidine/α-thio-uridine 5-methyl-uridine5-methyl-cytidine/5-methyl-uridine 5-iodo-cytidine5-methyl-cytidine/pseudouridine about 25% of cytosines are5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridinesare 2-thio-uridine about 50% of uridines are 5-methyl-cytidine/about 50%of uridines are 2-thio-uridine N4-acetyl-cytidine/5-iodo-uridineN4-acetyl-cytidine/N1-methyl-pseudouridineN4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridineN4-acetyl-cytidine/pseudouridine about 50% of cytosines areN4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidineN4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridineN4-acetyl-cytidine/2-thio-uridine about 50% of cytosines areN4-acetyl-cytidine/about 50% of uridines are 2-thio-uridinepseudoisocytidine/about 50% of uridines are N1-methyl- pseudouridine andabout 50% of uridines are pseudouridine pseudoisocytidine/about 25% ofuridines are N1-methyl- pseudouridine and about 25% of uridines arepseudouridine (e.g., 25% N1-methyl-pseudouridine/75% pseudouridine)about 50% of the cytosines are α-thio-cytidine

Certain modified nucleotides and nucleotide combinations have beenexplored by the current inventors. These findings are described in U.S.Provisional Application No. 61/404,413, filed on Oct. 1, 2010, entitledEngineered Nucleic Acids and Methods of Use Thereof, U.S. patentapplication Ser. No. 13/251,840, filed on Oct. 3, 2011, entitledModified Nucleotides, and Nucleic Acids, and Uses Thereof, nowabandoned, U.S. patent application Ser. No. 13/481,127, filed on May 25,2012, entitled Modified Nucleotides, and Nucleic Acids, and UsesThereof, International Patent Publication No WO2012045075, filed on Oct.3, 2011, entitled Modified Nucleosides, Nucleotides, And Nucleic Acids,and Uses Thereof, U.S. Patent Publication No US20120237975 filed on Oct.3, 2011, entitled Engineered Nucleic Acids and Method of Use Thereof,and International Patent Publication No WO2012045082, which areincorporated by reference in their entireties.

Further examples of modified nucleotide combinations are provided belowin Table 15. These combinations of modified nucleotides can be used toform the nucleic acids of the invention.

TABLE 15 Chemical Modifications Modified Nucleotide Modified NucleotideCombination modified cytidine having one or modified cytidine with(b10)/pseudouridine more nucleobases of Formula (b10) modified cytidinewith (b10)/N1-methyl- pseudouridine modified cytidine with(b10)/5-methoxy- uridine modified cytidine with (b10)/5-methyl-uridinemodified cytidine with (b10)/5-bromo-uridine modified cytidine with(b10)/2-thio-uridine about 50% of cytidine substituted with modifiedcytidine (b10)/about 50% of uridines are 2-thio-uridine modifiedcytidine having one or modified cytidine with (b32)/pseudouridine morenucleobases of Formula (b32) modified cytidine with (b32)/N1-methyl-pseudouridine modified cytidine with (b32)/5-methoxy- uridine modifiedcytidine with (b32)/5-methyl-uridine modified cytidine with(b32)/5-bromo-uridine modified cytidine with (b32)/2-thio-uridine about50% of cytidine substituted with modified cytidine (b32)/about 50% ofuridines are 2-thio-uridine modified uridine having one or more modifieduridine with (b1)/N4-acetyl- nucleobases of Formula (b1) cytidinemodified uridine with (b1)/5-methyl-cytidine modified uridine having oneor more modified uridine with (b8)/N4-acetyl- nucleobases of Formula(b8) cytidine modified uridine with (b8)/5-methyl-cytidine modifieduridine having one or more modified uridine with (b28)/N4-acetyl-nucleobases of Formula (b28) cytidine modified uridine with(b28)/5-methyl- cytidine modified uridine having one or more modifieduridine with (b29)/N4-acetyl- nucleobases of Formula (b29) cytidinemodified uridine with (b29)/5-methyl- cytidine modified uridine havingone or more modified uridine with (b30)/N4-acetyl- nucleobases ofFormula (b30) cytidine modified uridine with (b30)/5-methyl- cytidine

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14), (b24), (b25), or (b32)-(b35) (e.g., atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, orabout 100% of, e.g., a compound of Formula (b10) or (b32)).

In some embodiments, at least 25% of the uracils are replaced by acompound of Formula (b1)-(b9), (b21)-(b23), or (b28)-(b31) (e.g., atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, orabout 100% of, e.g., a compound of Formula (b1), (b8), (b28), (b29), or(b30)).

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14), (b24), (b25), or (b32)-(b35) (e.g.Formula (b10) or (b32)), and at least 25% of the uracils are replaced bya compound of Formula (b1)-(b9), (b21)-(b23), or (b28)-(b31) (e.g.Formula (b1), (b8), (b28), (b29), or (b30)) (e.g., at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%).

Modifications Including Linker and a Payload

Payload

The methods and compositions described herein are useful for deliveringa payload to a biological target. The payload can be used, e.g., forlabeling (e.g., a detectable agent such as a fluorophore), or fortherapeutic purposes (e.g., a cytotoxin or other therapeutic agent).

Payload: Therapeutic Agents

In some embodiments the payload is a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat.No. 5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499,5,846,545) and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, Samarium 153 and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, CC-1065, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

Payload: Detectable Agents

Examples of detectable substances include various organic smallmolecules, inorganic compounds, nanoparticles, enzymes or enzymesubstrates, fluorescent materials, luminescent materials, bioluminescentmaterials, chemiluminescent materials, radioactive materials, andcontrast agents. Such optically-detectable labels include for example,without limitation, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonicacid; acridine and derivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′ tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);Cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine. In some embodiments,the detectable label is a fluorescent dye, such as Cy5 and Cy3.

Examples luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin.

Examples of suitable radioactive material include ¹⁸F, ⁶⁷Ga, ^(81m)KR,⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl, ¹²⁵I, ³⁵S, ¹⁴C, or ³H, ^(99m)Tc (e.g.,as pertechnetate (technetate(VII), TcO₄ ⁻) either directly orindirectly, or other radioisotope detectable by direct counting ofradioemission or by scintillation counting.

In addition, contrast agents, e.g., contrast agents for MRI or NMR, forX-ray CT, Raman imaging, optical coherence tomography, absorptionimaging, ultrasound imaging, or thermal imaging can be used. Exemplarycontrast agents include gold (e.g., gold nanoparticles), gadolinium(e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide(SPIO), monocrystalline iron oxide nanoparticles (MIONs), and ultrasmallsuperparamagnetic iron oxide (USPIO)), manganese chelates (e.g.,Mn-DPDP), barium sulfate, iodinated contrast media (iohexyl),microbubbles, or perfluorocarbons can also be used.

In some embodiments, the detectable agent is a non-detectable pre-cursorthat becomes detectable upon activation. Examples include fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE (VisEn Medical)).

When the compounds are enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, theenzymatic label is detected by determination of conversion of anappropriate substrate to product.

In vitro assays in which these compositions can be used include enzymelinked immunosorbent assays (ELISAs), immunoprecipitations,immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA),and Western blot analysis.

Labels other than those described herein are contemplated by the presentdisclosure, including other optically-detectable labels. Labels can beattached to the modified nucleotide of the present disclosure at anyposition using standard chemistries such that the label can be removedfrom the incorporated base upon cleavage of the cleavable linker.

Payload: Cell Penetrating Payloads

In some embodiments, the modified nucleotides and modified nucleic acidscan also include a payload that can be a cell penetrating moiety oragent that enhances intracellular delivery of the compositions. Forexample, the compositions can include a cell-penetrating peptidesequence that facilitates delivery to the intracellular space, e.g.,HIV-derived TAT peptide, penetratins, transportans, or hCT derivedcell-penetrating peptides, see, e.g., Caron et al., (2001) Mol Ther.3(3):310-8; Langel, Cell-Penetrating Peptides: Processes andApplications (CRC Press, Boca Raton Fla. 2002); El-Andaloussi et al.,(2005) Curr Pharm Des. 11(28):3597-611; and Deshayes et al., (2005) CellMol Life Sci. 62(16):1839-49. The compositions can also be formulated toinclude a cell penetrating agent, e.g., liposomes, which enhancedelivery of the compositions to the intracellular space.

Payload: Biological Targets

The modified nucleotides and modified nucleic acids described herein canbe used to deliver a payload to any biological target for which aspecific ligand exists or can be generated. The ligand can bind to thebiological target either covalently or non-covalently.

Exemplary biological targets include biopolymers, e.g., antibodies,nucleic acids such as RNA and DNA, proteins, enzymes; exemplary proteinsinclude enzymes, receptors, and ion channels. In some embodiments thetarget is a tissue- or cell-type specific marker, e.g., a protein thatis expressed specifically on a selected tissue or cell type. In someembodiments, the target is a receptor, such as, but not limited to,plasma membrane receptors and nuclear receptors; more specific examplesinclude G-protein-coupled receptors, cell pore proteins, transporterproteins, surface-expressed antibodies, HLA proteins, MHC proteins andgrowth factor receptors.

Synthesis of Modified Nucleotides

The modified nucleosides and nucleotides disclosed herein can beprepared from readily available starting materials using the followinggeneral methods and procedures. It is understood that where typical orpreferred process conditions (i.e., reaction temperatures, times, moleratios of reactants, solvents, pressures, etc.) are given; other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry(e.g., UV-visible), or mass spectrometry, or by chromatography such ashigh performance liquid chromatography (HPLC) or thin layerchromatography.

Preparation of modified nucleosides and nucleotides can involve theprotection and deprotection of various chemical groups. The need forprotection and deprotection, and the selection of appropriate protectinggroups can be readily determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in Greene, etal., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons,1991, which is incorporated herein by reference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents, which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of modified nucleosides and nucleotidescan be carried out by any of numerous methods known in the art. Anexample method includes fractional recrystallization using a “chiralresolving acid” which is an optically active, salt-forming organic acid.Suitable resolving agents for fractional recrystallization methods are,for example, optically active acids, such as the D and L forms oftartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelicacid, malic acid, lactic acid or the various optically activecamphorsulfonic acids. Resolution of racemic mixtures can also becarried out by elution on a column packed with an optically activeresolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elutionsolvent composition can be determined by one skilled in the art.

Length

Generally, the length of a modified mRNA of the present invention isgreater than 30 nucleotides in length. In another embodiment, the RNAmolecule is greater than 35 nucleotides in length. In anotherembodiment, the length is at least 40 nucleotides. In anotherembodiment, the length is at least 45 nucleotides. In anotherembodiment, the length is at least 55 nucleotides. In anotherembodiment, the length is at least 60 nucleotides. In anotherembodiment, the length is at least 60 nucleotides. In anotherembodiment, the length is at least 80 nucleotides. In anotherembodiment, the length is at least 90 nucleotides. In anotherembodiment, the length is at least 100 nucleotides. In anotherembodiment, the length is at least 120 nucleotides. In anotherembodiment, the length is at least 140 nucleotides. In anotherembodiment, the length is at least 160 nucleotides. In anotherembodiment, the length is at least 180 nucleotides. In anotherembodiment, the length is at least 200 nucleotides. In anotherembodiment, the length is at least 250 nucleotides. In anotherembodiment, the length is at least 300 nucleotides. In anotherembodiment, the length is at least 350 nucleotides. In anotherembodiment, the length is at least 400 nucleotides. In anotherembodiment, the length is at least 450 nucleotides. In anotherembodiment, the length is at least 500 nucleotides. In anotherembodiment, the length is at least 600 nucleotides. In anotherembodiment, the length is at least 700 nucleotides. In anotherembodiment, the length is at least 800 nucleotides. In anotherembodiment, the length is at least 900 nucleotides. In anotherembodiment, the length is at least 1000 nucleotides. In anotherembodiment, the length is at least 1100 nucleotides. In anotherembodiment, the length is at least 1200 nucleotides. In anotherembodiment, the length is at least 1300 nucleotides. In anotherembodiment, the length is at least 1400 nucleotides. In anotherembodiment, the length is at least 1500 nucleotides. In anotherembodiment, the length is at least 1600 nucleotides. In anotherembodiment, the length is at least 1800 nucleotides. In anotherembodiment, the length is at least 2000 nucleotides. In anotherembodiment, the length is at least 2500 nucleotides. In anotherembodiment, the length is at least 3000 nucleotides. In anotherembodiment, the length is at least 4000 nucleotides. In anotherembodiment, the length is at least 5000 nucleotides, or greater than5000 nucleotides. In another embodiment, the length is at least 5000nucleotides, or greater than 6000 nucleotides. In another embodiment,the length is at least 7000 nucleotides, or greater than 7000nucleotides. In another embodiment, the length is at least 8000nucleotides, or greater than 8000 nucleotides. In another embodiment,the length is at least 9000 nucleotides, or greater than 9000nucleotides. In another embodiment, the length is at least 10,000nucleotides, or greater than 10,000 nucleotides.

Use of Modified RNAs

Prevention or Reduction of Innate Cellular Immune Response Activation

The term “innate immune response” includes a cellular response toexogenous single stranded nucleic acids, generally of viral or bacterialorigin, which involves the induction of cytokine expression and release,particularly the interferons, and cell death. Protein synthesis is alsoreduced during the innate cellular immune response. While it isadvantageous to eliminate the innate immune response in a cell, theinvention provides modified mRNAs that substantially reduce the immuneresponse, including interferon signaling, without entirely eliminatingsuch a response. In some embodiments, the immune response is reduced by10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greaterthan 99.9% as compared to the immune response induced by a correspondingunmodified nucleic acid. Such a reduction can be measured by expressionor activity level of Type 1 interferons or the expression ofinterferon-regulated genes such as the toll-like receptors (e.g., TLR7and TLR8). Reduction of innate immune response can also be measured bydecreased cell death following one or more administrations of modifiedRNAs to a cell population; e.g., cell death is 10%, 25%, 50%, 75%, 85%,90%, 95%, or over 95% less than the cell death frequency observed with acorresponding unmodified nucleic acid. Moreover, cell death may affectfewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than0.01% of cells contacted with the modified nucleic acids.

The invention provides for the repeated introduction (e.g.,transfection) of modified nucleic acids into a target cell population,e.g., in vitro, ex vivo, or in vivo. The step of contacting the cellpopulation may be repeated one or more times (such as two, three, four,five or more than five times). In some embodiments, the step ofcontacting the cell population with the modified nucleic acids isrepeated a number of times sufficient such that a predeterminedefficiency of protein translation in the cell population is achieved.Given the reduced cytotoxicity of the target cell population provided bythe nucleic acid modifications, such repeated transfections areachievable in a diverse array of cell types.

Major Groove Interacting Partners

As described herein, the phrase “major groove interacting partner”refers to RNA recognition receptors that detect and respond to RNAligands through interactions, e.g. binding, with the major groove faceof a nucleotide or nucleic acid. As such, RNA ligands comprisingmodified nucleotides or nucleic acids such as the modified RNAs asdescribed herein decrease interactions with major groove bindingpartners, and therefore decrease an innate immune response.

Example major groove interacting, e.g. binding, partners include, butare not limited to the following nucleases and helicases. Withinmembranes, TLRs (Toll-like Receptors) 3, 7, and 8 can respond to single-and double-stranded RNAs. Within the cytoplasm, members of thesuperfamily 2 class of DEX(D/H) helicases and ATPases can sense RNAs toinitiate antiviral responses. These helicases include the RIG-I(retinoic acid-inducible gene I) and MDA5 (melanomadifferentiation-associated gene 5). Other examples include laboratory ofgenetics and physiology 2 (LGP2), HIN-200 domain containing proteins, orHelicase-domain containing proteins.

RNA Binding Proteins

In some embodiments of the present invention, RNA binding proteins areprovided. RNA binding proteins may be provided as proteins and/or asnucleic acids encoding such proteins. RNA binding proteins play amultitude of roles in regulating RNA stability and protein translation.A/U rich elements in the 3′ UTR of mRNAs leads to formation of secondarystructures which are bound by A/U Rich Binding Proteins (AREBPs)resulting in increased or decreased mRNA stability (Fan, X. C. et al.,Overexpression of HuR, a nuclear-cytoplasmic shuttling protein,increases the in vivo stability of ARE-containing mRNAs. EMBO J. 1998Jun. 15; 17(12):3448-60). HuR is a stabilizing AREBP. To increase thestability of the mRNA of interest, an mRNA encoding HuR can beco-transfected or co-injected along with the mRNA of interest into thecells or into the tissue. These proteins can also be tethered to themRNA of interest in vitro and then administered to the cells together.Poly A tail binding protein, PABP interacts with eukaryotic translationinitiation factor eIF4G to stimulate translational initiation.Co-administration of mRNAs encoding these RBPs along with the mRNA drugand/or tethering these proteins to the mRNA drug in vitro andadministering the protein-bound mRNA into the cells can increase thetranslational efficiency of the mRNA. The same concept can be extendedto co-administration of mRNA along with mRNAs encoding varioustranslation factors and facilitators as well as with the proteinsthemselves to influence RNA stability and/or translational efficiency.

Polypeptide Variants

Provided are nucleic acids that encode variant polypeptides, which havea certain identity with a reference polypeptide sequence. The term“identity” as known in the art, refers to a relationship between thesequences of two or more peptides, as determined by comparing thesequences. In the art, “identity” also means the degree of sequencerelatedness between peptides, as determined by the number of matchesbetween strings of two or more amino acid residues.

“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related peptides can be readily calculated by known methods.Such methods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

In some embodiments, the polypeptide variant has the same or a similaractivity as the reference polypeptide. Alternatively, the variant has analtered activity (e.g., increased or decreased) relative to a referencepolypeptide. Generally, variants of a particular polynucleotide orpolypeptide of the invention will have at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to that particular referencepolynucleotide or polypeptide as determined by sequence alignmentprograms and parameters described herein and known to those skilled inthe art.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of this invention. For example, provided herein is anyprotein fragment of a reference protein (meaning a polypeptide sequenceat least one amino acid residue shorter than a reference polypeptidesequence but otherwise identical) 10, 20, 30, 40, 50, 60, 70, 80, 90,100 or greater than 100 amino acids in length In another example, anyprotein that includes a stretch of about 20, about 30, about 40, about50, or about 100 amino acids which are about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, or about 100% identical toany of the sequences described herein can be utilized in accordance withthe invention. In certain embodiments, a protein sequence to be utilizedin accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore mutations as shown in any of the sequences provided or referencedherein.

Polypeptide Libraries

Also provided are polynucleotide libraries containing nucleosidemodifications, wherein the polynucleotides individually contain a firstnucleic acid sequence encoding a polypeptide, such as an antibody,protein binding partner, scaffold protein, and other polypeptides knownin the art. Preferably, the polynucleotides are mRNA in a form suitablefor direct introduction into a target cell host, which in turnsynthesizes the encoded polypeptide.

In certain embodiments, multiple variants of a protein, each withdifferent amino acid modification(s), are produced and tested todetermine the best variant in terms of pharmacokinetics, stability,biocompatibility, and/or biological activity, or a biophysical propertysuch as expression level. Such a library may contain 10, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or over 10⁹ possible variants (includingsubstitutions, deletions of one or more residues, and insertion of oneor more residues).

Polypeptide-Nucleic Acid Complexes

Proper protein translation involves the physical aggregation of a numberof polypeptides and nucleic acids associated with the mRNA. Provided bythe invention are complexes containing conjugates of protein and nucleicacids, containing a translatable mRNA having one or more nucleosidemodifications (e.g., at least two different nucleoside modifications)and one or more polypeptides bound to the mRNA. Generally, the proteinsare provided in an amount effective to prevent or reduce an innateimmune response of a cell into which the complex is introduced.

Targeting Moieties

In embodiments of the invention, modified nucleic acids are provided toexpress a protein-binding partner or a receptor on the surface of thecell, which functions to target the cell to a specific tissue space orto interact with a specific moiety, either in vivo or in vitro. Suitableprotein-binding partners include antibodies and functional fragmentsthereof, scaffold proteins, or peptides. Additionally, modified nucleicacids can be employed to direct the synthesis and extracellularlocalization of lipids, carbohydrates, or other biological moieties.

As described herein, a useful feature of the modified nucleic acids ofthe invention is the capacity to reduce the innate immune response of acell to an exogenous nucleic acid. Provided are methods for performingthe titration, reduction or elimination of the immune response in a cellor a population of cells. In some embodiments, the cell is contactedwith a first composition that contains a first dose of a first exogenousnucleic acid including a translatable region and at least one nucleosidemodification, and the level of the innate immune response of the cell tothe first exogenous nucleic acid is determined. Subsequently, the cellis contacted with a second composition, which includes a second dose ofthe first exogenous nucleic acid, the second dose containing a lesseramount of the first exogenous nucleic acid as compared to the firstdose.

Alternatively, the cell is contacted with a first dose of a secondexogenous nucleic acid. The second exogenous nucleic acid may containone or more modified nucleosides, which may be the same or differentfrom the first exogenous nucleic acid or, alternatively, the secondexogenous nucleic acid may not contain modified nucleosides. The stepsof contacting the cell with the first composition and/or the secondcomposition may be repeated one or more times.

Additionally, efficiency of protein production (e.g., proteintranslation) in the cell is optionally determined, and the cell may bere-transfected with the first and/or second composition repeatedly untila target protein production efficiency is achieved.

In one embodiment, the 3′ end of the modified nucleic acids describedherein may include a sequence for targeting the modified nucleic acid toa desired location within the cell such as, but not limited to,microvesicles within a cell. The sequence for targeting may be “zipcode-like” in its function as it can be recognized by the cellularmachinery that can traffic molecules to various places within the cell.Non-limiting examples of sequences for targeting nucleic acids aredescribed in International Patent Publication No. WO2013109713, thecontents of which are herein incorporated by reference in its entirety.Zip-code like sequences and miR-1289 have been shown by Bolukbasi et al.to enrich mRNA in microvesicles (Mol. Ther. Nuc. Acid 2012 1, e10; thecontents of which are herein incorporated by reference in its entirety)as both zipcodes and microRNA have a role in post-transcriptionalregulation of mRNA.

In one embodiment, the sequence for targeting the modified nucleic acidis SEQ ID NO: 22, SEQ ID NO: 38 or a concatomer of at least one SEQ IDNO: 22 and at least one SEQ ID NO: 38 as described in InternationalPatent Publication No. WO2013109713, the contents of which are hereinincorporated by reference in its entirety.

Vaccines

As described herein, provided are mRNAs having sequences that aresubstantially not translatable. Such mRNA is effective as a vaccine whenadministered to a mammalian subject.

Also provided are modified nucleic acids that contain one or morenoncoding regions. Such modified nucleic acids are generally nottranslated, but are capable of binding to and sequestering one or moretranslational machinery component such as a ribosomal protein or atransfer RNA (tRNA), thereby effectively reducing protein expression inthe cell. The modified nucleic acid may contain a small nucleolar RNA(sno-RNA), micro RNA (miRNA), small interfering RNA (siRNA) orPiwi-interacting RNA (piRNA).

Additionally, certain modified nucleosides, or combinations thereof,when introduced into modified nucleic acids activate the innate immuneresponse. Such activating modified nucleic acids, e.g., modified RNAs,are useful as adjuvants when combined with polypeptide or othervaccines. In certain embodiments, the activated modified mRNAs contain atranslatable region which encodes for a polypeptide sequence useful as avaccine, thus providing the ability to be a self-adjuvant.

Therapeutic Agents

The modified nucleic acids (modified RNAs) and the proteins translatedfrom the modified nucleic acids described herein can be used astherapeutic agents. For example, a modified nucleic acid describedherein can be administered to a subject, wherein the modified nucleicacid is translated in vivo to produce a therapeutic peptide in thesubject. Provided are compositions, methods, kits, and reagents fortreatment or prevention of disease or conditions in humans and othermammals. The active therapeutic agents of the invention include modifiednucleic acids, cells containing modified nucleic acids or polypeptidestranslated from the modified nucleic acids, polypeptides translated frommodified nucleic acids, and cells contacted with cells containingmodified nucleic acids or polypeptides translated from the modifiednucleic acids.

In certain embodiments, provided are combination therapeutics containingone or more modified nucleic acids containing translatable regions thatencode for a protein or proteins that boost a mammalian subject'simmunity along with a protein that induces antibody-dependent cellulartoxitity. For example, provided are therapeutics containing one or morenucleic acids that encode trastuzumab and granulocyte-colony stimulatingfactor (G-CSF). In particular, such combination therapeutics are usefulin Her2+ breast cancer patients who develop induced resistance totrastuzumab. (See, e.g., Albrecht, Immunotherapy. 2(6):795-8 (2010)).

Provided are methods of inducing translation of a recombinantpolypeptide in a cell population using the modified nucleic acidsdescribed herein. Such translation can be in vivo, ex vivo, in culture,or in vitro. The cell population is contacted with an effective amountof a composition containing a nucleic acid that has at least onenucleoside modification, and a translatable region encoding therecombinant polypeptide. The population is contacted under conditionssuch that the nucleic acid is localized into one or more cells of thecell population and the recombinant polypeptide is translated in thecell from the nucleic acid.

An effective amount of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the nucleic acid (e.g., size, and extent ofmodified nucleosides), and other determinants. In general, an effectiveamount of the composition provides efficient protein production in thecell, preferably more efficient than a composition containing acorresponding unmodified nucleic acid. Increased efficiency may bedemonstrated by increased cell transfection (i.e., the percentage ofcells transfected with the nucleic acid), increased protein translationfrom the nucleic acid, decreased nucleic acid degradation (asdemonstrated, e.g., by increased duration of protein translation from amodified nucleic acid), or reduced innate immune response of the hostcell.

Aspects of the invention are directed to methods of inducing in vivotranslation of a recombinant polypeptide in a mammalian subject in needthereof. Therein, an effective amount of a composition containing anucleic acid that has at least one nucleoside modification and atranslatable region encoding the recombinant polypeptide is administeredto the subject using the delivery methods described herein. The nucleicacid is provided in an amount and under other conditions such that thenucleic acid is localized into a cell of the subject and the recombinantpolypeptide is translated in the cell from the nucleic acid. The cell inwhich the nucleic acid is localized, or the tissue in which the cell ispresent, may be targeted with one or more than one rounds of nucleicacid administration.

Other aspects of the invention relate to transplantation of cellscontaining modified nucleic acids to a mammalian subject. Administrationof cells to mammalian subjects is known to those of ordinary skill inthe art, such as local implantation (e.g., topical or subcutaneousadministration), organ delivery or systemic injection (e.g., intravenousinjection or inhalation), as is the formulation of cells inpharmaceutically acceptable carrier. Compositions containing modifiednucleic acids are formulated for administration intramuscularly,transarterially, intraocularly, vaginally, rectally, intraperitoneally,intravenously, intranasally, subcutaneously, endoscopically,transdermally, or intrathecally. In some embodiments, the composition isformulated for extended release.

The subject to whom the therapeutic agent is administered suffers fromor is at risk of developing a disease, disorder, or deleteriouscondition. Provided are methods of identifying, diagnosing, andclassifying subjects on these bases, which may include clinicaldiagnosis, biomarker levels, genome-wide association studies (GWAS), andother methods known in the art.

In certain embodiments, the administered modified nucleic acid directsproduction of one or more recombinant polypeptides that provide afunctional activity which is substantially absent in the cell in whichthe recombinant polypeptide is translated. For example, the missingfunctional activity may be enzymatic, structural, or gene regulatory innature. In related embodiments, the administered modified nucleic aciddirects production of one or more recombinant polypeptides thatincreases (e.g., synergistically) a functional activity which is presentbut substantially deficient in the cell in which the recombinantpolypeptide is translated.

In other embodiments, the administered modified nucleic acid directsproduction of one or more recombinant polypeptides that replace apolypeptide (or multiple polypeptides) that is substantially absent inthe cell in which the recombinant polypeptide is translated. Suchabsence may be due to genetic mutation of the encoding gene orregulatory pathway thereof. In some embodiments, the recombinantpolypeptide increases the level of an endogenous protein in the cell toa desirable level; such an increase may bring the level of theendogenous protein from a subnormal level to a normal level, or from anormal level to a super-normal level.

Alternatively, the recombinant polypeptide functions to antagonize theactivity of an endogenous protein present in, on the surface of, orsecreted from the cell. Usually, the activity of the endogenous proteinis deleterious to the subject, for example, do to mutation of theendogenous protein resulting in altered activity or localization.Additionally, the recombinant polypeptide antagonizes, directly orindirectly, the activity of a biological moiety present in, on thesurface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a nucleic acid, a carbohydrate, aprotein toxin such as shiga and tetanus toxins, or a small moleculetoxin such as botulinum, cholera, and diphtheria toxins. Additionally,the antagonized biological molecule may be an endogenous protein thatexhibits an undesirable activity, such as a cytotoxic or cytostaticactivity. The recombinant proteins described herein are engineered forlocalization within the cell, potentially within a specific compartmentsuch as the nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

Therapeutics

Provided are methods for treating or preventing a symptom of diseasescharacterized by missing or aberrant protein activity, by replacing themissing protein activity or overcoming the aberrant protein activity.Because of the rapid initiation of protein production followingintroduction of modified mRNAs, as compared to viral DNA vectors, thecompounds of the present invention are particularly advantageous intreating acute diseases such as sepsis, stroke, and myocardialinfarction. Moreover, the lack of transcriptional regulation of themodified mRNAs of the invention is advantageous in that accuratetitration of protein production is achievable.

In some embodiments, modified mRNAs and their encoded polypeptides inaccordance with the present invention may be used for therapeuticpurposes. In some embodiments, modified mRNAs and their encodedpolypeptides in accordance with the present invention may be used fortreatment of any of a variety of diseases, disorders, and/or conditions,including but not limited to one or more of the following: autoimmunedisorders (e.g. diabetes, lupus, multiple sclerosis, psoriasis,rheumatoid arthritis); inflammatory disorders (e.g. arthritis, pelvicinflammatory disease); infectious diseases (e.g. viral infections (e.g.,HIV, HCV, RSV), bacterial infections, fungal infections, sepsis);neurological disorders (e.g. Alzheimer's disease, Huntington's disease;autism; Duchenne muscular dystrophy); cardiovascular disorders (e.g.atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders,angiogenic disorders such as macular degeneration); proliferativedisorders (e.g. cancer, benign neoplasms); respiratory disorders (e.g.chronic obstructive pulmonary disease); digestive disorders (e.g.inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g.fibromyalgia, arthritis); endocrine, metabolic, and nutritionaldisorders (e.g. diabetes, osteoporosis); urological disorders (e.g.renal disease); psychological disorders (e.g. depression,schizophrenia); skin disorders (e.g. wounds, eczema); blood andlymphatic disorders (e.g. anemia, hemophilia); etc.

Diseases characterized by dysfunctional or aberrant protein activityinclude cystic fibrosis, sickle cell anemia, epidermolysis bullosa,amyotrophic lateral sclerosis, and glucose-6-phosphate dehydrogenasedeficiency. The present invention provides a method for treating suchconditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the modified nucleic acids providedherein, wherein the modified nucleic acids encode for a protein thatantagonizes or otherwise overcomes the aberrant protein activity presentin the cell of the subject. Specific examples of a dysfunctional proteinare the missense mutation variants of the cystic fibrosis transmembraneconductance regulator (CFTR) gene, which produce a dysfunctional proteinvariant of CFTR protein, which causes cystic fibrosis.

Diseases characterized by missing (or substantially diminished such thatproper protein function does not occur) protein activity include cysticfibrosis, Niemann-Pick type C, β thalassemia major, Duchenne musculardystrophy, Hurler Syndrome, Hunter Syndrome, and Hemophilia A. Suchproteins may not be present, or are essentially non-functional. Thepresent invention provides a method for treating such conditions ordiseases in a subject by introducing nucleic acid or cell-basedtherapeutics containing the modified nucleic acids provided herein,wherein the modified nucleic acids encode for a protein that replacesthe protein activity missing from the target cells of the subject.Specific examples of a dysfunctional protein are the nonsense mutationvariants of the cystic fibrosis transmembrane conductance regulator(CFTR) gene, which produce a nonfunctional protein variant of CFTRprotein, which causes cystic fibrosis.

Thus, provided are methods of treating cystic fibrosis in a mammaliansubject by contacting a cell of the subject with a modified nucleic acidhaving a translatable region that encodes a functional CFTR polypeptide,under conditions such that an effective amount of the CTFR polypeptideis present in the cell. Preferred target cells are epithelial,endothelial and mesothelial cells, such as the lung, and methods ofadministration are determined in view of the target tissue; i.e., forlung delivery, the RNA molecules are formulated for administration byinhalation.

In another embodiment, the present invention provides a method fortreating hyperlipidemia in a subject, by introducing into a cellpopulation of the subject with a modified mRNA molecule encodingSortilin, a protein recently characterized by genomic studies, therebyameliorating the hyperlipidemia in a subject. The SORT1 gene encodes atrans-Golgi network (TGN) transmembrane protein called Sortilin. Geneticstudies have shown that one of five individuals has a single nucleotidepolymorphism, rs12740374, in the 1p13 locus of the SORT1 gene thatpredisposes them to having low levels of low-density lipoprotein (LDL)and very-low-density lipoprotein (VLDL). Each copy of the minor allele,present in about 30% of people, alters LDL cholesterol by 8 mg/dL, whiletwo copies of the minor allele, present in about 5% of the population,lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have alsobeen shown to have a 40% decreased risk of myocardial infarction.Functional in vivo studies in mice describes that overexpression ofSORT1 in mouse liver tissue led to significantly lower LDL-cholesterollevels, as much as 80% lower, and that silencing SORT1 increased LDLcholesterol approximately 200% (Musunuru K et al. From noncoding variantto phenotype via SORT1 at the 1p13 cholesterol locus. Nature 2010; 466:714-721, herein incorporated by reference in its entirety.).

Modulation of Cell Fate

Provided are methods of inducing an alteration in cell fate in a targetmammalian cell. The target mammalian cell may be a precursor cell andthe alteration may involve driving differentiation into a lineage, orblocking such differentiation. Alternatively, the target mammalian cellmay be a differentiated cell, and the cell fate alteration includesdriving de-differentiation into a pluripotent precursor cell, orblocking such de-differentiation, such as the dedifferentiation ofcancer cells into cancer stem cells. In situations where a change incell fate is desired, effective amounts of mRNAs encoding a cell fateinductive polypeptide is introduced into a target cell under conditionssuch that an alteration in cell fate is induced. In some embodiments,the modified mRNAs are useful to reprogram a subpopulation of cells froma first phenotype to a second phenotype. Such a reprogramming may betemporary or permanent.

Optionally, the reprogramming induces a target cell to adopt anintermediate phenotype.

Additionally, the methods of the present invention are particularlyuseful to generate induced pluripotent stem cells (iPS cells) because ofthe high efficiency of transfection, the ability to re-transfect cells,and the tenability of the amount of recombinant polypeptides produced inthe target cells. Further, the use of iPS cells generated using themethods described herein is expected to have a reduced incidence ofteratoma formation.

Also provided are methods of reducing cellular differentiation in atarget cell population. For example, a target cell population containingone or more precursor cell types is contacted with a composition havingan effective amount of a modified mRNA encoding a polypeptide, underconditions such that the polypeptide is translated and reduces thedifferentiation of the precursor cell. In non-limiting embodiments, thetarget cell population contains injured tissue in a mammalian subject ortissue affected by a surgical procedure. The precursor cell is, e.g., astromal precursor cell, a neural precursor cell, or a mesenchymalprecursor cell.

In a specific embodiment, provided are modified nucleic acids thatencode one or more differentiation factors Gata4, Mef2c and Tbx4. ThesemRNA-generated factors are introduced into fibroblasts and drive thereprogramming into cardiomyocytes. Such a reprogramming can be performedin vivo, by contacting an mRNA-containing patch or other material todamaged cardiac tissue to facilitate cardiac regeneration. Such aprocess promotes cardiomyocyte genesis as opposed to fibrosis.

Targeting of Pathogenic Organisms; Purification of Biological Materials

Provided herein are methods for targeting pathogenic microorganisms,such as bacteria, yeast, protozoa, helminthes and the like, usingmodified mRNAs that encode cytostatic or cytotoxic polypeptides.Preferably the mRNA introduced into the target pathogenic organismcontains modified nucleosides or other nucleic acid sequencemodifications that the mRNA is translated exclusively, orpreferentially, in the target pathogenic organism, to reduce possibleoff-target effects of the therapeutic. Such methods are useful forremoving pathogenic organisms from biological material, including blood,semen, eggs, and transplant materials including embryos, tissues, andorgans.

Targeting Diseased Cells

Provided herein are methods for targeting pathogenic or diseased cells,particularly cancer cells, using modified mRNAs that encode cytostaticor cytotoxic polypeptides. Preferably the mRNA introduced into thetarget pathogenic cell contains modified nucleosides or other nucleicacid sequence modifications that the mRNA is translated exclusively, orpreferentially, in the target pathogenic cell, to reduce possibleoff-target effects of the therapeutic. Alternatively, the inventionprovides targeting moieties that are capable of targeting the modifiedmRNAs to preferentially bind to and enter the target pathogenic cell.

Protein Production

The methods provided herein are useful for enhancing protein productyield in a cell culture process. In a cell culture containing aplurality of host cells, introduction of the modified mRNAs describedherein results in increased protein production efficiency relative to acorresponding unmodified nucleic acid. Such increased protein productionefficiency can be demonstrated, e.g., by showing increased celltransfection, increased protein translation from the nucleic acid,decreased nucleic acid degradation, and/or reduced innate immuneresponse of the host cell. Protein production can be measured by ELISA,and protein activity can be measured by various functional assays knownin the art. The protein production may be generated in a continuous or afed-batch mammalian process.

Additionally, it is useful to optimize the expression of a specificpolypeptide in a cell line or collection of cell lines of potentialinterest, particularly an engineered protein such as a protein variantof a reference protein having a known activity. In one embodiment,provided is a method of optimizing expression of an engineered proteinin a target cell, by providing a plurality of target cell types, andindependently contacting with each of the plurality of target cell typesa modified mRNA encoding an engineered polypeptide. Additionally,culture conditions may be altered to increase protein productionefficiency. Subsequently, the presence and/or level of the engineeredpolypeptide in the plurality of target cell types is detected and/orquantitated, allowing for the optimization of an engineeredpolypeptide's expression by selection of an efficient target cell andcell culture conditions relating thereto. Such methods are particularlyuseful when the engineered polypeptide contains one or morepost-translational modifications or has substantial tertiary structure,situations which often complicate efficient protein production.

Gene Silencing

The modified mRNAs described herein are useful to silence (i.e., preventor substantially reduce) expression of one or more target genes in acell population. A modified mRNA encoding a polypeptide capable ofdirecting sequence-specific histone H3 methylation is introduced intothe cells in the population under conditions such that the polypeptideis translated and reduces gene transcription of a target gene viahistone H3 methylation and subsequent heterochromatin formation. In someembodiments, the silencing mechanism is performed on a cell populationpresent in a mammalian subject. By way of non-limiting example, a usefultarget gene is a mutated Janus Kinase-2 family member, wherein themammalian subject expresses the mutant target gene suffers from amyeloproliferative disease resulting from aberrant kinase activity.

Co-administration of modified mRNAs and siRNAs are also provided herein.As demonstrated in yeast, sequence-specific trans silencing is aneffective mechanism for altering cell function. Fission yeast requiretwo RNAi complexes for siRNA-mediated heterochromatin assembly: theRNA-induced transcriptional silencing (RITS) complex and theRNA-directed RNA polymerase complex (RDRC) (Motamedi et al. Cell 2004,119, 789-802). In fission yeast, the RITS complex contains the siRNAbinding Argonaute family protein Ago1, a chromodomain protein Chp1, andTas3. The fission yeast RDRC complex is composed of an RNA-dependent RNAPolymerase Rdp1, a putative RNA helicase Hrr1, and a polyA polymerasefamily protein Cid12. These two complexes require the Dicer ribonucleaseand Clr4 histone H3 methyltransferase for activity. Together, Ago1 bindssiRNA molecules generated through Dicer-mediated cleavage of Rdp1co-transcriptionally generated dsRNA transcripts and allows for thesequence-specific direct association of Chp1, Tas3, Hrr1, and Clr4 toregions of DNA destined for methylation and histone modification andsubsequent compaction into transcriptionally silenced heterochromatin.While this mechanism functions in cis- with centromeric regions of DNA,sequence-specific trans silencing is possible through co-transfectionwith double-stranded siRNAs for specific regions of DNA and concomitantRNAi-directed silencing of the siRNA ribonuclease Eri1 (Buhler et al.Cell 2006, 125, 873-886, herein incorporated by reference in itsentirety.).

Modulation of Biological Pathways

The rapid translation of modified mRNAs introduced into cells provides adesirable mechanism of modulating target biological pathways. Suchmodulation includes antagonism or agonism of a given pathway. In oneembodiment, a method is provided for antagonizing a biological pathwayin a cell by contacting the cell with an effective amount of acomposition comprising a modified nucleic acid encoding a recombinantpolypeptide, under conditions such that the nucleic acid is localizedinto the cell and the recombinant polypeptide is capable of beingtranslated in the cell from the nucleic acid, wherein the recombinantpolypeptide inhibits the activity of a polypeptide functional in thebiological pathway. Exemplary biological pathways are those defective inan autoimmune or inflammatory disorder such as multiple sclerosis,rheumatoid arthritis, psoriasis, lupus erythematosus, ankylosingspondylitis colitis, or Crohn's disease; in particular, antagonism ofthe IL-12 and IL-23 signaling pathways are of particular utility. (SeeKikly K, Liu L, Na S, Sedgwick J D (2006) Curr. Opin. Immunol. 18 (6):670-5, herein incorporated by reference in its entirety.).

Further, provided are modified nucleic acids encoding an antagonist forchemokine receptors; chemokine receptors CXCR-4 and CCR-5 are requiredfor, e.g., HIV entry into host cells (Arenzana-Seisdedos F et al, (1996)Nature. October 3; 383(6599):400, herein incorporated by reference inits entirety.).

Alternatively, provided are methods of agonizing a biological pathway ina cell by contacting the cell with an effective amount of a modifiednucleic acid encoding a recombinant polypeptide under conditions suchthat the nucleic acid is localized into the cell and the recombinantpolypeptide is capable of being translated in the cell from the nucleicacid, and the recombinant polypeptide induces the activity of apolypeptide functional in the biological pathway. Exemplary agonizedbiological pathways include pathways that modulate cell fatedetermination. Such agonization is reversible or, alternatively,irreversible.

Cellular Nucleic Acid Delivery

Methods of the present invention enhance nucleic acid delivery into acell population, in vivo, ex vivo, or in culture. For example, a cellculture containing a plurality of host cells (e.g., eukaryotic cellssuch as yeast or mammalian cells) is contacted with a composition thatcontains an enhanced nucleic acid having at least one nucleosidemodification and, optionally, a translatable region. The compositionalso generally contains a transfection reagent or other compound thatincreases the efficiency of enhanced nucleic acid uptake into the hostcells. The enhanced nucleic acid exhibits enhanced retention in the cellpopulation, relative to a corresponding unmodified nucleic acid. Theretention of the enhanced nucleic acid is greater than the retention ofthe unmodified nucleic acid. In some embodiments, it is at least about50%, 75%, 90%, 95%, 100%, 150%, 200% or more than 200% greater than theretention of the unmodified nucleic acid. Such retention advantage maybe achieved by one round of transfection with the enhanced nucleic acid,or may be obtained following repeated rounds of transfection.

In some embodiments, the enhanced nucleic acid is delivered to a targetcell population with one or more additional nucleic acids. Such deliverymay be at the same time, or the enhanced nucleic acid is delivered priorto delivery of the one or more additional nucleic acids. The additionalone or more nucleic acids may be modified nucleic acids or unmodifiednucleic acids. It is understood that the initial presence of theenhanced nucleic acids does not substantially induce an innate immuneresponse of the cell population and, moreover, that the innate immuneresponse will not be activated by the later presence of the unmodifiednucleic acids. In this regard, the enhanced nucleic acid may not itselfcontain a translatable region, if the protein desired to be present inthe target cell population is translated from the unmodified nucleicacids.

IV. Pharmaceutical Compositions

Formulation, Administration, Delivery and Dosing

The present invention provides polynucleotides, modified nucleic acid,enhanced modified RNA and ribonucleic acid compositions and complexes incombination with one or more pharmaceutically acceptable excipients.Pharmaceutical compositions may optionally comprise one or moreadditional active substances, e.g. therapeutically and/orprophylactically active substances. General considerations in theformulation and/or manufacture of pharmaceutical agents may be found,for example, in Remington: The Science and Practice of Pharmacy 21^(st)ed., Lippincott Williams & Wilkins, 2005 (incorporated herein byreference).

In one embodiment, provided are formulations containing an effectiveamount of a ribonucleic acid (e.g., an mRNA or a nucleic acid containingan mRNA) engineered to avoid an innate immune response of a cell intowhich the ribonucleic acid enters. The ribonucleic acid generallyincludes a nucleotide sequence encoding a polypeptide of interest.

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to a modified nucleic acid,an enhanced nucleic aicd or a ribonucleic acid to be delivered asdescribed herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient

Formulations

The polynucleotides, modified nucleic acid, enhanced modified RNA andribonucleic acid of the invention can be formulated using one or moreexcipients to: (1) increase stability; (2) increase cell transfection;(3) permit the sustained or delayed release (e.g., from a depotformulation of the modified nucleic acids, enhanced modified RNA orribonucleic acids); (4) alter the biodistribution (e.g., target themodified nucleic acids, enhanced modified RNA or ribonucleic acids tospecific tissues or cell types); (5) increase the translation of encodedprotein in vivo; and/or (6) alter the release profile of encoded proteinin vivo. In addition to traditional excipients such as any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, excipients of thepresent invention can include, without limitation, lipidoids, liposomes,lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles,peptides, proteins, cells transfected with polynucleotides, modifiednucleic acid, enhanced modified RNA and ribonucleic acid (e.g., fortransplantation into a subject), hyaluronidase, nanoparticle mimics andcombinations thereof.

Accordingly, the formulations of the invention can include one or moreexcipients, each in an amount that together increases the stability ofthe polynucleotide, modified nucleic acid, enhanced modified RNA orribonucleic acid, increases cell transfection by the polynucleotides,modified nucleic acid, enhanced modified RNA or ribonucleic acid,increases the expression of polynucleotides, modified nucleic acid,enhanced modified RNA or ribonucleic acid encoded protein, and/or altersthe release profile of the polynucleotides, modified nucleic acid,enhanced modified RNA or ribonucleic acid encoded proteins. Further, thepolynucleotides, modified nucleic acid, enhanced modified RNA orribonucleic acid of the present invention may be formulated usingself-assembled nucleic acid nanoparticles.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

The polynucleotides, modified nucleic acid, enhanced modified RNA andribonucleic acid of the invention may be formulated for delivery in thetissues and/or organs of a subject. Organs may include, but are notlimited to, the heart, lung, brain, liver, basal ganglia, brain stemmedulla, midbrain, pons, cerebellum, cerebral cortex, hypothalamus, eye,pituitary, thyroid, parathyroid, esophagus, thymus, adrenal glands,appendix, bladder, gallbladder, intestines (e.g., large intestine andsmall intestine), kidney, pancreas, spleen, stomach, skin, prostate,testes, ovaries, uterus, adrenal glands, anus, bronchi, ears, esophagus,genitals, larynx (voice box), lymph nodes, meninges, mouth, nose,parathyroid glands, pituitary gland, rectum, salivary glands, spinalcord, thymus gland, tongue, trachea, ureters, urethra, colon. Tissuesmay include, but are not limited to, heart valves, bone, vein, middleear, muscle (cardiac, smooth or skeletal) cartilage, tendon orligaments. As a non-limiting example, the polynucleotides, modifiednucleic acid, enhanced modified RNA and ribonucleic acid may beformulated in a lipid nanoparticle and delivered to an organ such as,but not limited, to the liver, spleen, kidney or lung. In anothernon-limiting example, the polynucleotides, modified nucleic acids,enhanced modified RNA and ribonucleic acid may be formulated in a lipidnanoparticle comprising the cationic lipid DLin-KC2-DMA and delivered toan organ such as, but not limited to, the liver, spleen, kidney or lung.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient may generally be equal to the dosage of theactive ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage including, but not limited to,one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient.

In some embodiments, the modified mRNA formulations described herein maycontain at least one modified mRNA. The formulations may contain 1, 2,3, 4 or 5 modified mRNA. In one embodiment the formulation may containmodified mRNA encoding proteins selected from categories such as, butnot limited to, human proteins, veterinary proteins, bacterial proteins,biological proteins, antibodies, immunogenic proteins, therapeuticpeptides and proteins, secreted proteins, plasma membrane proteins,cytoplasmic and cytoskeletal proteins, intrancellular membrane boundproteins, nuclear proteins, proteins associated with human diseaseand/or proteins associated with non-human diseases. In one embodiment,the formulation contains at least three modified mRNA encoding proteins.In one embodiment, the formulation contains at least five modified mRNAencoding proteins.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro,Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference). The use of a conventional excipient medium may becontemplated within the scope of the present disclosure, except insofaras any conventional excipient medium may be incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition.

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter biological reaction such as, but not limited to,inflammation or may increase the biological effect of the modified mRNAdelivered to mammals.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, surface active agents and/or emulsifiers, preservatives,buffering agents, lubricating agents, and/or oils. Such excipients mayoptionally be included in the pharmaceutical formulations of theinvention

Lipidoid

The synthesis of lipidoids has been extensively described andformulations containing these compounds are particularly suited fordelivery of polynucleotides, modified nucleic acids, enhanced modifiedRNA and ribonucleic acids (see Mahon et al., Bioconjug Chem. 201021:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc etal., Nat. Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad SciUSA. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-3001; all of which are incorporated herein in theirentireties).

While these lipidoids have been used to effectively deliver doublestranded small interfering RNA molecules in rodents and non-humanprimates (see Akinc et al., Nat. Biotechnol. 2008 26:561-569;Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920;Akinc et al., Mol. Ther. 2009 17:872-879; Love et al., Proc Natl AcadSci USA. 2010 107:1864-1869; Leuschner et al., Nat. Biotechnol. 201129:1005-1010; all of which is incorporated herein in their entirety),the present disclosure describes their formulation and use in deliveringsingle stranded polynucleotide, modified nucleic acids, enhancedmodified RNA and ribonucleic acids. Complexes, micelles, liposomes orparticles can be prepared containing these lipidoids and therefore, canresult in an effective delivery of the polynucleotides, modified nucleicacids, enhanced modified RNA and ribonucleic acids, as judged by theproduction of an encoded protein, following the injection of a lipidoidformulation via localized and/or systemic routes of administration.Lipidoid complexes of polynucleotides, modified nucleic acids, enhancedmodified RNA and ribonucleic acids can be administered by various meansincluding, but not limited to, intravenous, intramuscular, orsubcutaneous routes.

In vivo delivery of nucleic acids may be affected by many parameters,including, but not limited to, the formulation composition, nature ofparticle PEGylation, degree of loading, oligonucleotide to lipid ratio,and biophysical parameters such as particle size (Akinc et al., MolTher. 2009 17:872-879; herein incorporated by reference in itsentirety). As an example, small changes in the anchor chain length ofpoly(ethylene glycol) (PEG) lipids may result in significant effects onin vivo efficacy. Formulations with the different lipidoids, including,but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry,401:61 (2010)), C12-200 (including derivatives and variants), and MD1,can be tested for in vivo activity.

The lipidoid referred to herein as “98N12-5” is disclosed by Akinc etal., Mol Ther. 2009 17:872-879 and is incorporated by reference in itsentirety.

The lipidoid referred to herein as “C12-200” is disclosed by Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 and Liu and Huang,Molecular Therapy. 2010 669-670; both of which are herein incorporatedby reference in their entirety. The lipidoid formulations can includeparticles comprising either 3 or 4 or more components in addition topolynucleotide, modified nucleic acids, enhanced modified RNA andribonucleic acids. As an example, formulations with certain lipidoids,include, but are not limited to, 98N12-5 and may contain 42% lipidoid,48% cholesterol and 10% PEG (C₁₋₄ alkyl chain length). As anotherexample, formulations with certain lipidoids, include, but are notlimited to, C12-200 and may contain 50% lipidoid, 10%disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.

In one embodiment, a modified nucleic acids, enhanced modified RNA orribonucleic acids formulated with a lipidoid for systemic intravenousadministration can target the liver. For example, a final optimizedintravenous formulation using modified nucleic acids, enhanced modifiedRNA or ribonucleic acids, and comprising a lipid molar composition of42% 98N12-5, 48% cholesterol, and 10% PEG-lipid with a final weightratio of about 7.5 to 1 total lipid to polynucleotide, modified nucleicacids, enhanced modified RNA or ribonucleic acids, and a C₁₋₄ alkylchain length on the PEG lipid, with a mean particle size of roughly50-60 nm, can result in the distribution of the formulation to begreater than 90% to the liver. (see, Akinc et al., Mol. Ther. 200917:872-879; herein incorporated in its entirety). In another example, anintravenous formulation using a C12-200 (see U.S. provisionalapplication 61/175,770 and published international applicationWO2010129709, each of which is herein incorporated by reference in theirentirety) lipidoid may have a molar ratio of 50/10/38.5/1.5 ofC12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG, with a weightratio of 7 to 1 total lipid to polynucleotide, modified nucleic acids,enhanced modified RNA or ribonucleic acids, and a mean particle size of80 nm may be effective to deliver polynucleotides, modified nucleicacids, enhanced modified RNA or ribonucleic acids to hepatocytes (see,Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869 hereinincorporated by reference). In another embodiment, an MD1lipidoid-containing formulation may be used to effectively deliverpolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids to hepatocytes in vivo. The characteristics ofoptimized lipidoid formulations for intramuscular or subcutaneous routesmay vary significantly depending on the target cell type and the abilityof formulations to diffuse through the extracellular matrix into theblood stream. While a particle size of less than 150 nm may be desiredfor effective hepatocyte delivery due to the size of the endothelialfenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879 hereinincorporated by reference), use of a lipidoid-formulatedpolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids to deliver the formulation to other cells typesincluding, but not limited to, endothelial cells, myeloid cells, andmuscle cells may not be similarly size-limited. Use of lipidoidformulations to deliver siRNA in vivo to other non-hepatocyte cells suchas myeloid cells and endothelium has been reported (see Akinc et al.,Nat. Biotechnol. 2008 26:561-569; Leuschner et al., Nat. Biotechnol.2011 29:1005-1010; Cho et al. Adv. Funct. Mater. 2009 19:3112-3118;8^(th) International Judah Folkman Conference, Cambridge, Mass. Oct.8-9, 2010 herein incorporated by reference in its entirety). Effectivedelivery to myeloid cells, such as monocytes, lipidoid formulations mayhave a similar component molar ratio. Different ratios of lipidoids andother components including, but not limited to, disteroylphosphatidylcholine, cholesterol and PEG-DMG, may be used to optimize theformulation of the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids for delivery to different cell typesincluding, but not limited to, hepatocytes, myeloid cells, muscle cells,etc. For example, the component molar ratio may include, but is notlimited to, 50% C12-200, 10% disteroylphosphatidyl choline, 38.5%cholesterol, and %1.5 PEG-DMG (see Leuschner et al., Nat Biotechnol 201129:1005-1010; herein incorporated by reference in its entirety). The useof lipidoid formulations for the localized delivery of nucleic acids tocells (such as, but not limited to, adipose cells and muscle cells) viaeither subcutaneous or intramuscular delivery, may not require all ofthe formulation components desired for systemic delivery, and as suchmay comprise only the lipidoid and the polynucleotides, modified nucleicacids, enhanced modified RNA or ribonucleic acids.

Combinations of different lipidoids may be used to improve the efficacyof polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids directed protein production as the lipidoids may beable to increase cell transfection by the polynucleotides, modifiednucleic acid, or modified nucleic acids, enhanced modified RNA orribonucleic acids; and/or increase the translation of encoded protein(see Whitehead et al., Mol. Ther. 2011, 19:1688-1694, hereinincorporated by reference in its entirety).

Liposomes, Lipoplexes, and Lipid Nanoparticles

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention can be formulated using one or moreliposomes, lipoplexes, or lipid nanoparticles. In one embodiment,pharmaceutical compositions of polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids include liposomes. Liposomesare artificially-prepared vesicles which may primarily be composed of alipid bilayer and may be used as a delivery vehicle for theadministration of nutrients and pharmaceutical formulations. Liposomescan be of different sizes such as, but not limited to, a multilamellarvesicle (MLV) which may be hundreds of nanometers in diameter and maycontain a series of concentric bilayers separated by narrow aqueouscompartments, a small unicellular vesicle (SUV) which may be smallerthan 50 nm in diameter, and a large unilamellar vesicle (LUV) which maybe between 50 and 500 nm in diameter. Liposome design may include, butis not limited to, opsonins or ligands in order to improve theattachment of liposomes to unhealthy tissue or to activate events suchas, but not limited to, endocytosis. Liposomes may contain a low or ahigh pH in order to improve the delivery of the pharmaceuticalformulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.). Inone embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat. Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;all of which are incorporated herein in their entireties.) The originalmanufacture method by Wheeler et al. was a detergent dialysis method,which was later improved by Jeffs et al. and is referred to as thespontaneous vesicle formation method. The liposome formulations arecomposed of 3 to 4 lipid components in addition to the polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acids. Asan example a liposome can contain, but is not limited to, 55%cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG,and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described byJeffs et al. As another example, certain liposome formulations maycontain, but are not limited to, 48% cholesterol, 20% DSPC, 2%PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described byHeyes et al.

In one embodiment, pharmaceutical compositions may include liposomeswhich may be formed to deliver polynucleotides, modified nucleic acids,enhanced modified RNA and ribonucleic acids which may encode at leastone immunogen. The polynucleotides, modified nucleic acids, enhancedmodified RNA and ribonucleic acids may be encapsulated by the liposomeand/or it may be contained in an aqueous core which may then beencapsulated by the liposome (see International Pub. Nos. WO2012031046,WO2012031043, WO2012030901 and WO2012006378; each of which is hereinincorporated by reference in their entirety). In anotherpolynucleotides, embodiment, the modified nucleic acids, enhancedmodified RNA and ribonucleic acids which may encode an immunogen may beformulated in a cationic oil-in-water emulsion where the emulsionparticle comprises an oil core and a cationic lipid which can interactwith the polynucleotides, modified nucleic acids, enhanced modified RNAand ribonucleic acids anchoring the molecule to the emulsion particle(see International Pub. No. WO2012006380). In yet another embodiment,the lipid formulation may include at least cationic lipid, a lipid whichmay enhance transfection and a least one lipid which contains ahydrophilic head group linked to a lipid moiety (International Pub. No.WO2011076807 and U.S. Pub. No. 20110200582; each of which is hereinincorporated by reference in their entirety). In another embodiment, thepolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids encoding an immunogen may be formulated in a lipidvesicle which may have crosslinks between functionalized lipid bilayers(see U.S. Pub. No. 20120177724, herein incorporated by reference in itsentirety).

In one embodiment, the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids may be formulated in a lipid vesiclewhich may have crosslinks between functionalized lipid bilayers.

In one embodiment, the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids may be formulated in alipid-polycation complex. The formation of the lipid-polycation complexmay be accomplished by methods known in the art and/or as described inU.S. Pub. No. 20120178702, herein incorporated by reference in itsentirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine. In another embodiment,the polynucleotide, modified nucleic acids, enhanced modified RNA orribonucleic acids may be formulated in a lipid-polycation complex whichmay further include a neutral lipid such as, but not limited to,cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176), the liposomeformulation was composed of 57.1% cationic lipid, 7.1%dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.As another example, changing the composition of the cationic lipid couldmore effectively deliver siRNA to various antigen presenting cells(Basha et al. Mol Ther. 2011 19:2186-2200; herein incorporated byreference in its entirety).

In some embodiments, the ratio of PEG in the LNP formulations may beincreased or decreased and/or the carbon chain length of the PEG lipidmay be modified from C14 to C18 to alter the pharmacokinetics and/orbiodistribution of the LNP formulations. As a non-limiting example, LNPformulations may contain 1-5% of the lipid molar ratio of PEG-c-DOMG ascompared to the cationic lipid, DSPC and cholesterol. In anotherembodiment the PEG-c-DOMG may be replaced with a PEG lipid such as, butnot limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethyleneglycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethyleneglycol). The cationic lipid may be selected from any lipid known in theart such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 andDLin-KC2-DMA.

In one embodiment, the cationic lipid may be selected from, but notlimited to, a cationic lipid described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865 and WO2008103276, U.S. Pat. Nos. 7,893,302 and 7,404,969 andUS Patent Publication No. US20100036115; each of which is hereinincorporated by reference in their entirety. In another embodiment, thecationic lipid may be selected from, but not limited to, formula Adescribed in International Publication Nos. WO2012040184, WO2011153120,WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259,WO2012054365 and WO2012044638; each of which is herein incorporated byreference in their entirety. In yet another embodiment, the cationiclipid may be selected from, but not limited to, formula CLI-CLXXIX ofInternational Publication No. WO2008103276, formula CLI-CLXXIX of U.S.Pat. No. 7,893,302, formula CLI-CLXXXXII of U.S. Pat. No. 7,404,969 andformula I-VI of US Patent Publication No. US20100036115; each of whichis herein incorporated by reference in their entirety. As a non-limitingexample, the cationic lipid may be selected from(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(1Z,19Z)—N5N˜dimethylpentacosa-16,19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13J16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-9-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z;19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine,(21Z,24Z)—N;N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimethylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—NJN-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20J23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21˜[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyH-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine(Compound 9);(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amineand (11E,20Z,23Z)—N;N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the cationic lipid may be synthesized by methodsknown in the art and/or as described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 andWO201021865; each of which is herein incorporated by reference in theirentirety.

In one embodiment, the LNP formulation may contain PEG-c-DOMG 3% lipidmolar ratio. In another embodiment, the LNP formulation may containPEG-c-DOMG 1.5% lipid molar ratio.

In one embodiment, the LNP formulation may contain PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethyleneglycol)-2000). In one embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art and at least one othercomponent. In another embodiment, the LNP formulation may containPEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.As a non-limiting example, the LNP formulation may contain PEG-DMG 2000,DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNPformulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol ina molar ratio of 2:40:10:48 (see Geall et al., Nonviral delivery ofself-amplifying RNA vaccines, PNAS 2012; PMID: 22908294).

In one embodiment, the LNP formulation may be formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276, each of which is herein incorporated by reference in theirentirety. As a non-limiting example, modified RNA described herein maybe encapsulated in LNP formulations as described in WO2011127255 and/orWO2008103276; each of which is herein incorporated by reference in theirentirety.

In one embodiment, LNP formulations described herein may comprise apolycationic composition. As a non-limiting example, the polycationiccomposition may be selected from formula 1-60 of US Patent PublicationNo. US20050222064; herein incorporated by reference in its entirety. Inanother embodiment, the LNP formulations comprising a polycationiccomposition may be used for the delivery of the modified RNA describedherein in vivo and/or in vitro.

In one embodiment, the LNP formulations described herein mayadditionally comprise a permeability enhancer molecule. Non-limitingpermeability enhancer molecules are described in US Patent PublicationNo. US20050222064; herein incorporated by reference in its entirety.

In one embodiment, the pharmaceutical compositions may be formulated inliposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutralDOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,siRNA delivery for ovarian cancer (Landen et al. Cancer Biology &Therapy 2006 5(12)1708-1713)) and hyaluronan-coated liposomes (QuietTherapeutics, Israel).

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In one embodiment, the internal ester linkage may be located on eitherside of the saturated carbon. Non-limiting examples of reLNPs include,

In one embodiment, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; each of which is herein incorporated byreference in their entirety). The polymer may encapsulate thenanospecies or partially encapsulate the nanospecies. The immunogen maybe a recombinant protein, a modified RNA described herein. In oneembodiment, the lipid nanoparticle may be formulated for use in avaccine such as, but not limited to, against a pathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosla tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; each of which is herein incorporated by reference in theirentirety). The transport of nanoparticles may be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT).

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. Non-limiting examples of specificpolymers include poly(caprolactone) (PCL), ethylene vinyl acetatepolymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA),poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA),poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA),poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer, and(poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol))triblock copolymer (see US Publication 20120121718 and US Publication20100003337; each of which is herein incorporated by reference in theirentirety). The co-polymer may be a polymer that is generally regarded assafe (GRAS) and the formation of the lipid nanoparticle may be in such away that no new chemical entities are created. For example, the lipidnanoparticle may comprise poloxamers coating PLGA nanoparticles withoutforming new chemical entities which are still able to rapidly penetratehuman mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; hereinincorporated by reference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, polynucleotides, modifiednucleic acids, enhanced modified RNA, ribonucleic acids, anionic protein(e.g., bovine serum albumin), surfactants (e.g., cationic surfactantssuch as for example dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g.,N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol,sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosinβ4 dornase alfa, neltenexine, erdosteine) and various DNases includingrhDNase. The surface altering agent may be embedded or enmeshed in theparticle's surface or disposed (e.g., by coating, adsorption, covalentlinkage, or other process) on the surface of the lipid nanoparticle.(see US Publication 20100215580 and US Publication 20080166414; each ofwhich is herein incorporated by reference in their entirety).

The mucus penetrating lipid nanoparticles may comprise at least onepolynucleotide, modified nucleic acids, enhanced modified RNA orribonucleic acids described herein. The modified nucleic acids, enhancedmodified RNA or ribonucleic acids may be encapsulated in the lipidnanoparticle and/or disposed on the surface of the particle. Thepolynucleotide, modified nucleic acids, enhanced modified RNA orribonucleic acids may be covalently coupled to the lipid nanoparticle.Formulations of mucus penetrating lipid nanoparticles may comprise aplurality of nanoparticles. Further, the formulations may containparticles which may interact with the mucus and alter the structuraland/or adhesive properties of the surrounding mucus to decreasemucoadhesion which may increase the delivery of the mucus penetratinglipid nanoparticles to the mucosal tissue.

In one embodiment, the polynucleotide, modified nucleic acids, enhancedmodified RNA or ribonucleic acids is formulated as a lipoplex, such as,without limitation, the ATUPLEX™ system, the DACC system, the DBTCsystem and other siRNA-lipoplex technology from Silence Therapeutics(London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.),and polyethylenimine (PEI) or protamine-based targeted and non-targeteddelivery of nucleic acids (Aleku et al. Cancer Res. 2008 68:9788-9798;Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al.,Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370;Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al.Microvasc Res 2010 80:286-293 Weide et al. J Immunother. 200932:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo ExpertOpin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother.34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al.,Proc Natl Acad Sci USA. 2007 6; 104:4095-4100; deFougerolles Hum GeneTher. 2008 19:125-132; all of which are incorporated herein by referencein its entirety).

In one embodiment such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and MC3-based lipid nanoparticle formulationswhich have been shown to bind to apolipoprotein E and promote bindingand uptake of these formulations into hepatocytes in vivo (Akinc et al.Mol Ther. 2010 18:1357-1364; herein incorporated by reference in itsentirety). Formulations can also be selectively targeted throughexpression of different ligands on their surface as exemplified by, butnot limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), andantibody targeted approaches (Kolhatkar et al., Curr Drug DiscovTechnol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 201116:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al.,Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al.,Biomacromolecules. 2011 12:2708-2714 Zhao et al., Expert Opin DrugDeliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364;Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al.,Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release.20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kimet al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther.2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer etal., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 201118:1127-1133; all of which are incorporated herein by reference in itsentirety).

In one embodiment, the polynucleotide, modified nucleic acids, enhancedmodified RNA or ribonucleic acids is formulated as a solid lipidnanoparticle. A solid lipid nanoparticle (SLN) may be spherical with anaverage diameter between 10 to 1000 nm. SLN possess a solid lipid corematrix that can solubilize lipophilic molecules and may be stabilizedwith surfactants and/or emulsifiers. In a further embodiment, the lipidnanoparticle may be a self-assembly lipid-polymer nanoparticle (seeZhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; herein incorporatedby reference in its entirety).

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotides, modified nucleic acids, enhanced modifiedRNA or ribonucleic acids directed protein production as theseformulations may be able to increase cell transfection by thepolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids; and/or increase the translation of encoded protein.One such example involves the use of lipid encapsulation to enable theeffective systemic delivery of polyplex plasmid DNA (Heyes et al., MolTher. 2007 15:713-720; herein incorporated by reference in itsentirety). The liposomes, lipoplexes, or lipid nanoparticles may also beused to increase the stability of the polynucleotides, modified nucleicacids, enhanced modified RNA or ribonucleic acids.

In one embodiment, the polynucleotide, modified nucleic acids, enhancedmodified RNA or ribonucleic acids of the present invention can beformulated for controlled release and/or targeted delivery. As usedherein, “controlled release” refers to a pharmaceutical composition orcompound release profile that conforms to a particular pattern ofrelease to effect a therapeutic outcome. In one embodiment, thepolynucleotide, modified nucleic acids, enhanced modified RNA orribonucleic acids may be encapsulated into a delivery agent describedherein and/or known in the art for controlled release and/or targeteddelivery. As used herein, the term “encapsulate” means to enclose,surround or encase. As it relates to the formulation of the compounds ofthe invention, encapsulation may be substantial, complete or partial.The term “substantially encapsulated” means that at least greater than50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than99.999% of the pharmaceutical composition or compound of the inventionmay be enclosed, surrounded or encased within the delivery agent.“Partially encapsulation” means that less than 10, 10, 20, 30, 40 50 orless of the pharmaceutical composition or compound of the invention maybe enclosed, surrounded or encased within the delivery agent.Advantageously, encapsulation may be determined by measuring the escapeor the activity of the pharmaceutical composition or compound of theinvention using fluorescence and/or electron micrograph. For example, atleast 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99,99.9, 99.99 or greater than 99.99% of the pharmaceutical composition orcompound of the invention are encapsulated in the delivery agent.

In another embodiment, the polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids may be encapsulated into alipid nanoparticle or a rapidly eliminating lipid nanoparticle and thelipid nanoparticles or a rapidly eliminating lipid nanoparticle may thenbe encapsulated into a polymer, hydrogel and/or surgical sealantdescribed herein and/or known in the art. As a non-limiting example, thepolymer, hydrogel or surgical sealant may be PLGA, ethylene vinylacetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua,Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgicalsealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.),TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

In one embodiment, the lipid nanoparticle may be encapsulated into anypolymer or hydrogel known in the art which may form a gel when injectedinto a subject. As another non-limiting example, the lipid nanoparticlemay be encapsulated into a polymer matrix which may be biodegradable.

In one embodiment, the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids formulation for controlled releaseand/or targeted delivery may also include at least one controlledrelease coating. Controlled release coatings include, but are notlimited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethyl cellulose, EUDRAGIT RED, EUDRAGIT RS® andcellulose derivatives such as ethylcellulose aqueous dispersions(AQUACOAT® and SURELEASE®).

In one embodiment, the controlled release and/or targeted deliveryformulation may comprise at least one degradable polyester which maycontain polycationic side chains. Degradeable polyesters include, butare not limited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters may include a PEG conjugation toform a PEGylated polymer.

In one embodiment, the modified nucleic acids, enhanced modified RNA orribonucleic acids of the present invention may be encapsulated in atherapeutic nanoparticle. Therapeutic nanoparticles may be formulated bymethods described herein and known in the art such as, but not limitedto, International Pub Nos. WO2010005740, WO2010030763, WO2010005721,WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645,US20100087337, US20100068285, US20110274759, US20100068286, and U.S.Pat. No. 8,206,747; each of which is herein incorporated by reference intheir entirety. In another embodiment, therapeutic polymer nanoparticlesmay be identified by the methods described in US Pub No. US20120140790,herein incorporated by reference in its entirety.

In one embodiment, the therapeutic nanoparticle may be formulated forsustained release. As used herein, “sustained release” refers to apharmaceutical composition or compound that conforms to a release rateover a specific period of time. The period of time may include, but isnot limited to, hours, days, weeks, months and years. As a non-limitingexample, the sustained release nanoparticle may comprise a polymer and atherapeutic agent such as, but not limited to, the polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acids ofthe present invention (see International Pub No. 2010075072 and US PubNo. US20100216804 and US20110217377, each of which is hereinincorporated by reference in their entirety).

In one embodiment, the therapeutic nanoparticles may be formulated to betarget specific. As a non-limiting example, the therapeuticnanoparticles may include a corticosteroid (see International Pub. No.WO2011084518). In one embodiment, the therapeutic nanoparticles may beformulated to be cancer specific. As a non-limiting example, thetherapeutic nanoparticles may be formulated in nanoparticles describedin International Pub No. WO2008121949, WO2010005726, WO2010005725,WO2011084521 and US Pub No. US20100069426, US20120004293 andUS20100104655, each of which is herein incorporated by reference intheir entirety.

In one embodiment, the nanoparticles of the present invention maycomprise a polymeric matrix. As a non-limiting example, the nanoparticlemay comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In one embodiment, the diblock copolymer may include PEG in combinationwith a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblockcopolymer. As a non-limiting example the therapeutic nanoparticlecomprises a PLGA-PEG block copolymer (see US Pub. No. US20120004293 andU.S. Pat. No. 8,236,330, each of which is herein incorporated byreference in their entirety). In another non-limiting example, thetherapeutic nanoparticle is a stealth nanoparticle comprising a diblockcopolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968,herein incorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle may comprise at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In one embodiment, the therapeutic nanoparticles may comprise at leastone cationic polymer described herein and/or known in the art.

In one embodiment, the therapeutic nanoparticles may comprise at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers and combinationsthereof.

In one embodiment, the therapeutic nanoparticles may comprise at leastone degradable polyester which may contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters may include a PEG conjugation to form a PEGylated polymer.

In another embodiment, the therapeutic nanoparticle may include aconjugation of at least one targeting ligand.

In one embodiment, the therapeutic nanoparticle may be formulated in anaqueous solution which may be used to target cancer (see InternationalPub No. WO2011084513 and US Pub No. US20110294717, each of which isherein incorporated by reference in their entirety).

In one embodiment, the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids may be encapsulated in, linked toand/or associated with synthetic nanocarriers. The syntheticnanocarriers may be formulated using methods known in the art and/ordescribed herein. As a non-limiting example, the synthetic nanocarriersmay be formulated by the methods described in International Pub Nos.WO2010005740, WO2010030763 and US Pub. Nos. US20110262491, US20100104645and US20100087337, each of which is herein incorporated by reference intheir entirety. In another embodiment, the synthetic nanocarrierformulations may be lyophilized by methods described in InternationalPub. No. WO2011072218 and U.S. Pat. No. 8,211,473; each of which isherein incorporated by reference in their entirety.

In one embodiment, the synthetic nanocarriers may contain reactivegroups to release the modified nucleic acids, enhanced modified RNA orribonucleic acids described herein (see International Pub. No.WO20120952552 and US Pub No. US20120171229, each of which is hereinincorporated by reference in their entirety).

In one embodiment, the synthetic nanocarriers may contain animmunostimulatory agent to enhance the immune response from delivery ofthe synthetic nanocarrier. As a non-limiting example, the syntheticnanocarrier may comprise a Th1 immunostimulatory agent which may enhancea Th1-based response of the immune system (see International Pub No.WO2010123569 and US Pub. No. US20110223201, each of which is hereinincorporated by reference in its entirety).

In one embodiment, the synthetic nanocarriers may be formulated fortargeted release. In one embodiment, the synthetic nanocarrier isformulated to release the polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids at a specified pH and/orafter a desired time interval. As a non-limiting example, the syntheticnanoparticle may be formulated to release the modified nucleic acids,enhanced modified RNA or ribonucleic acids after 24 hours and/or at a pHof 4.5 (see International Pub. Nos. WO2010138193 and WO2010138194 and USPub Nos. US20110020388 and US20110027217, each of which is hereinincorporated by reference in their entirety).

In one embodiment, the synthetic nanocarriers may be formulated forcontrolled and/or sustained release of the polynucleotides, modifiednucleic acids, enhanced modified RNA or ribonucleic acids describedherein. As a non-limiting example, the synthetic nanocarriers forsustained release may be formulated by methods known in the art,described herein and/or as described in International Pub No.WO2010138192 and US Pub No. 20100303850, each of which is hereinincorporated by reference in their entirety.

In one embodiment, the synthetic nanocarrier may be formulated for useas a vaccine. In one embodiment, the synthetic nanocarrier mayencapsulate at least one modified nucleic acids, enhanced modified RNAor ribonucleic acids which encodes at least one antigen. As anon-limiting example, the synthetic nanocarrier may include at least oneantigen and an excipient for a vaccine dosage form (see InternationalPub No. WO2011150264 and US Pub No. US20110293723, each of which isherein incorporated by reference in their entirety). As anothernon-limiting example, a vaccine dosage form may include at least twosynthetic nanocarriers with the same or different antigens and anexcipient (see International Pub No. WO2011150249 and US Pub No.US20110293701, each of which is herein incorporated by reference intheir entirety). The vaccine dosage form may be selected by methodsdescribed herein, known in the art and/or described in International PubNo. WO2011150258 and US Pub No. US20120027806, each of which is hereinincorporated by reference in their entirety).

In one embodiment, the synthetic nanocarrier may comprise at least onepolynucleotide, modified nucleic acids, enhanced modified RNA orribonucleic acids which encodes at least one adjuvant. In anotherembodiment, the synthetic nanocarrier may comprise at least one modifiednucleic acids, enhanced modified RNA or ribonucleic acids and anadjuvant. As a non-limiting example, the synthetic nanocarriercomprising and adjuvant may be formulated by the methods described inInternational Pub No. WO2011150240 and US Pub No. US20110293700, each ofwhich is herein incorporated by reference in its entirety.

In one embodiment, the synthetic nanocarrier may encapsulate at leastone polynucleotide, modified nucleic acids, enhanced modified RNA orribonucleic acids which encodes a peptide, fragment or region from avirus. As a non-limiting example, the synthetic nanocarrier may include,but is not limited to, the nanocarriers described in International PubNo. WO2012024621, WO201202629, WO2012024632 and US Pub No.US20120064110, US20120058153 and US20120058154, each of which is hereinincorporated by reference in their entirety.

Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention can be formulated using naturaland/or synthetic polymers. Non-limiting examples of polymers which maybe used for delivery include, but are not limited to, DynamicPOLYCONJUGATE™ formulations from MIRUS® Bio (Madison, Wis.) and RocheMadison (Madison, Wis.), PHASERX™ polymer formulations such as, withoutlimitation, SMARTT POLYMER TECHNOLOGY™ (Seattle, Wash.), DMRI/DOPE,poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan,cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimersand poly(lactic-co-glycolic acid) (PLGA) polymers, RONDEL™(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (ArrowheadResearch Corporation, Pasadena, Calif.) and pH responsive co-blockpolymers such as, but not limited to, PHASERX™ (Seattle, Wash.).

A non-limiting example of PLGA formulations include, but are not limitedto, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolvingPLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueoussolvent and leuprolide. Once injected, the PLGA and leuprolide peptideprecipitates into the subcutaneous space).

Many of these polymer approaches have demonstrated efficacy indelivering oligonucleotides in vivo into the cell cytoplasm (reviewed indeFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated byreference in its entirety). Two polymer approaches that have yieldedrobust in vivo delivery of nucleic acids, in this case with smallinterfering RNA (siRNA), are dynamic polyconjugates andcyclodextrin-based nanoparticles. The first of these delivery approachesuses dynamic polyconjugates and has been shown in vivo in mice toeffectively deliver siRNA and silence endogenous target mRNA inhepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887). This particular approach is a multicomponent polymersystem whose key features include a membrane-active polymer to whichnucleic acid, in this case siRNA, is covalently coupled via a disulfidebond and where both PEG (for charge masking) and N-acetylgalactosamine(for hepatocyte targeting) groups are linked via pH-sensitive bonds(Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887). Onbinding to the hepatocyte and entry into the endosome, the polymercomplex disassembles in the low-pH environment, with the polymerexposing its positive charge, leading to endosomal escape andcytoplasmic release of the siRNA from the polymer. Through replacementof the N-acetylgalactosamine group with a mannose group, it was shownone could alter targeting from asialoglycoprotein receptor-expressinghepatocytes to sinusoidal endothelium and Kupffer cells. Another polymerapproach involves using transferrin-targeted cyclodextrin-containingpolycation nanoparticles. These nanoparticles have demonstrated targetedsilencing of the EWS-FLI™ gene product in transferrinreceptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et al.,Cancer Res. 2005 65: 8984-8982) and siRNA formulated in thesenanoparticles was well tolerated in non-human primates (Heidel et al.,Proc Natl Acad Sci USA 2007 104:5715-21). Both of these deliverystrategies incorporate rational approaches using both targeted deliveryand endosomal escape mechanisms.

The polymer formulation can permit the sustained or delayed release ofpolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids (e.g., following intramuscular or subcutaneousinjection). The altered release profile for the polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acids canresult in, for example, translation of an encoded protein over anextended period of time. The polymer formulation may also be used toincrease the stability of the polynucleotide, modified nucleic acids,enhanced modified RNA or ribonucleic acids. Biodegradable polymers havebeen previously used to protect nucleic acids other than modifiednucleic acids, enhanced modified RNA or ribonucleic acids fromdegradation and been shown to result in sustained release of payloads invivo (Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887;Sullivan et al., Expert Opin Drug Deliv. 2010 7:1433-1446; Convertine etal., Biomacromolecules. 2010 Oct. 1; Chu et al., Acc Chem Res. 2012 Jan.13; Manganiello et al., Biomaterials. 2012 33:2301-2309; Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Singha et al., Nucleic Acid Ther.2011 2:133-147; deFougerolles Hum Gene Ther. 2008 19:125-132; Schaffertand Wagner, Gene Ther. 2008 16:1131-1138; Chaturvedi et al., Expert OpinDrug Deliv. 2011 8:1455-1468; Davis, Mol Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; herein incorporated by reference in itsentirety).

In one embodiment, the pharmaceutical compositions may be sustainedrelease formulations. In a further embodiment, the sustained releaseformulations may be for subcutaneous delivery. Sustained releaseformulations may include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

As a non-limiting example modified mRNA may be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradeable,biocompatible polymers which are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine device; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C. PEG-based surgical sealants comprise two syntheticPEG components mixed in a delivery device which can be prepared in oneminute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE®and natural polymers are capable of in-situ gelation at the site ofadministration. They have been shown to interact with protein andpeptide therapeutic candidates through ionic interaction to provide astabilizing effect.

Polymer formulations can also be selectively targeted through expressionof different ligands as exemplified by, but not limited by, folate,transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; each of which is herein incorporated byreference in its entirety).

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention may be formulated with or in apolymeric compound. The polymer may include at least one polymer suchas, but not limited to, polyethenes, polyethylene glycol (PEG),poly(1-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer,biodegradable cationic lipopolymer, polyethyleneimine (PEI),cross-linked branched poly(alkylene imines), a polyamine derivative, amodified poloxamer, a biodegradable polymer, biodegradable blockcopolymer, biodegradable random copolymer, biodegradable polyestercopolymer, biodegradable polyester block copolymer, biodegradablepolyester block random copolymer, linear biodegradable copolymer,poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradablecross-linked cationic multi-block copolymers, polycarbonates,polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), acrylic polymers, amine-containingpolymers or combinations thereof.

As a non-limiting example, the polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids of the invention may beformulated with the polymeric compound of PEG grafted with PLL asdescribed in U.S. Pat. No. 6,177,274 herein incorporated by reference inits entirety. The formulation may be used for transfecting cells invitro or for in vivo delivery of the modified nucleic acids, enhancedmodified RNA or ribonucleic acids. In another example, thepolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids may be suspended in a solution or medium with acationic polymer, in a dry pharmaceutical composition or in a solutionthat is capable of being dried as described in U.S. Pub. Nos.20090042829 and 20090042825 each of which are herein incorporated byreference in their entireties.

As another non-limiting example the polynucleotides, modified nucleicacids, enhanced modified RNA or ribonucleic acids of the invention maybe formulated with a PLGA-PEG block copolymer (see US Pub. No.US20120004293 and U.S. Pat. No. 8,236,330, each of which are hereinincorporated by reference in their entireties). As a non-limitingexample, the polynucleotides, modified nucleic acids, enhanced modifiedRNA or ribonucleic acids of the invention may be formulated with adiblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No.8,246,968, herein incorporated by reference in its entirety).

A polyamine derivative may be used to deliver nucleic acids or to treatand/or prevent a disease or to be included in an implantable orinjectable device (U.S. Pub. No. 20100260817 herein incorporated byreference in its entirety). As a non-limiting example, a pharmaceuticalcomposition may include the modified nucleic acids, enhanced modifiedRNA or ribonucleic acids and the polyamine derivative described in U.S.Pub. No. 20100260817 (the contents of which are incorporated herein byreference in its entirety).

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention may be formulated with at least oneacrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In one embodiment, the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids of the present invention may beformulated with at least one polymer described in InternationalPublication Nos. WO2011115862, WO2012082574 and WO2012068187, each ofwhich are herein incorporated by reference in their entireties. Inanother embodiment, the polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids of the present invention maybe formulated with a polymer of formula Z as described in WO2011115862,herein incorporated by reference in its entirety. In yet anotherembodiment, the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids may be formulated with a polymer offormula Z, Z′ or Z″ as described in WO2012082574 or WO2012068187, eachof which are herein incorporated by reference in their entireties. Thepolymers formulated with the modified RNA of the present invention maybe synthesized by the methods described in WO2012082574 or WO2012068187,each of which are herein incorporated by reference in their entireties.

Formulations of polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids of the invention may include at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers or combinations thereof.

For example, the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids of the invention may be formulated ina pharmaceutical compound including a poly(alkylene imine), abiodegradable cationic lipopolymer, a biodegradable block copolymer, abiodegradable polymer, or a biodegradable random copolymer, abiodegradable polyester block copolymer, a biodegradable polyesterpolymer, a biodegradable polyester random copolymer, a linearbiodegradable copolymer, PAGA, a biodegradable cross-linked cationicmulti-block copolymer or combinations thereof. The biodegradablecationic lipopolymer may be made by methods known in the art and/ordescribed in U.S. Pat. No. 6,696,038, U.S. App. Nos. 20030073619 and20040142474 each of which is herein incorporated by reference in theirentireties. The poly(alkylene imine) may be made using methods known inthe art and/or as described in U.S. Pub. No. 20100004315, hereinincorporated by reference in its entirety. The biodegradabale polymer,biodegradable block copolymer, the biodegradable random copolymer,biodegradable polyester block copolymer, biodegradable polyesterpolymer, or biodegradable polyester random copolymer may be made usingmethods known in the art and/or as described in U.S. Pat. Nos. 6,517,869and 6,267,987, the contents of which are each incorporated herein byreference in its entirety. The linear biodegradable copolymer may bemade using methods known in the art and/or as described in U.S. Pat. No.6,652,886. The PAGA polymer may be made using methods known in the artand/or as described in U.S. Pat. No. 6,217,912 herein incorporated byreference in its entirety. The PAGA polymer may be copolymerized to forma copolymer or block copolymer with polymers such as but not limited to,poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines,polylactides and poly(lactide-co-glycolides). The biodegradablecross-linked cationic multi-block copolymers may be made my methodsknown in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S.Pub. No. 2012009145 each of which are herein incorporated by referencein their entireties. For example, the multi-block copolymers may besynthesized using linear polyethyleneimine (LPEI) blocks which havedistinct patterns as compared to branched polyethyleneimines. Further,the composition or pharmaceutical composition may be made by the methodsknown in the art, described herein, or as described in U.S. Pub. No.20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912 each of which areherein incorporated by reference in their entireties.

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention may be formulated with at least onedegradable polyester which may contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters may include a PEG conjugation to form a PEGylated polymer.

In one embodiment, the polymers described herein may be conjugated to alipid-terminating PEG. As a non-limiting example, PLGA may be conjugatedto a lipid-terminating PEG forming PLGA-DSPE-PEG. As anothernon-limiting example, PEG conjugates for use with the present inventionare described in International Publication No. WO2008103276, hereinincorporated by reference in its entirety.

In one embodiment, the polynucleotides, modified RNA described hereinmay be conjugated with another compound. Non-limiting examples ofconjugates are described in U.S. Pat. Nos. 7,964,578 and 7,833,992, eachof which are herein incorporated by reference in their entireties. Inanother embodiment, modified RNA of the present invention may beconjugated with conjugates of formula I-122 as described in U.S. Pat.Nos. 7,964,578 and 7,833,992, each of which are herein incorporated byreference in their entireties.

As described in U.S. Pub. No. 20100004313, herein incorporated byreference in its entirety, a gene delivery composition may include anucleotide sequence and a poloxamer. For example, the polynucleotide,modified nucleic acids, enhanced modified RNA or ribonucleic acids ofthe present invention may be used in a gene delivery composition withthe poloxamer described in U.S. Pub. No. 20100004313.

In one embodiment, the polymer formulation of the present invention maybe stabilized by contacting the polymer formulation, which may include acationic carrier, with a cationic lipopolymer which may be covalentlylinked to cholesterol and polyethylene glycol groups. The polymerformulation may be contacted with a cationic lipopolymer using themethods described in U.S. Pub. No. 20090042829 herein incorporated byreference in its entirety. The cationic carrier may include, but is notlimited to, polyethylenimine, poly(trimethylenimine),poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine,dideoxy-diamino-b-cyclodextrin, spermine, spermidine,poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine),poly(arginine), cationized gelatin, dendrimers, chitosan,1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),3B—[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl) diheptadecylamidoglycyl spermidine (DOGS),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) andcombinations thereof.

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention can also be formulated as ananoparticle using a combination of polymers, lipids, and/or otherbiodegradable agents, such as, but not limited to, calcium phosphate.Components may be combined in a core-shell, hybrid, and/orlayer-by-layer architecture, to allow for fine-tuning of thenanoparticle so to deliver the modified nucleic acids, enhanced modifiedRNA or ribonucleic acids may be enhanced (Wang et al., Nat Mater. 20065:791-796; Fuller et al., Biomaterials. 2008 29:1526-1532; DeKoker etal., Adv Drug Deliv Rev. 2011 63:748-761; Endres et al., Biomaterials.2011 32:7721-7731; Su et al., Mol Pharm. 2011 Jun. 6; 8(3):774-87;herein incorporated by reference in its entirety).

Biodegradable calcium phosphate nanoparticles in combination with lipidsand/or polymers have been shown to deliver polynucleotides, modifiednucleic acids, enhanced modified RNA or ribonucleic acids in vivo. Inone embodiment, a lipid coated calcium phosphate nanoparticle, which mayalso contain a targeting ligand such as anisamide, may be used todeliver the polynucleotides, modified nucleic acids, enhanced modifiedRNA or ribonucleic acids of the present invention. For example, toeffectively deliver siRNA in a mouse metastatic lung model a lipidcoated calcium phosphate nanoparticle was used (Li et al., J Contr Rel.2010 142: 416-421; Li et al., J Contr Rel. 2012 158:108-114; Yang etal., Mol. Ther. 2012 20:609-615). This delivery system combines both atargeted nanoparticle and a component to enhance the endosomal escape,calcium phosphate, in order to improve delivery of the siRNA.

In one embodiment, calcium phosphate with a PEG-polyanion blockcopolymer may be used to deliver polynucleotides, modified nucleicacids, enhanced modified RNA or ribonucleic acids (Kazikawa et al., JContr Rel. 2004 97:345-356; Kazikawa et al., J Contr Rel. 2006111:368-370).

In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle todeliver the polynucleotides, modified nucleic acids, enhanced modifiedRNA or ribonucleic acids of the present invention. ThePEG-charge-conversional polymer may improve upon the PEG-polyanion blockcopolymers by being cleaved into a polycation at acidic pH, thusenhancing endosomal escape.

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001). The complexation, delivery, and internalization of thepolymeric nanoparticles can be precisely controlled by altering thechemical composition in both the core and shell components of thenanoparticle. For example, the core-shell nanoparticles may efficientlydeliver siRNA to mouse hepatocytes after they covalently attachcholesterol to the nanoparticle.

In one embodiment, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer containing PEG may be used to deliveryof the polynucleotide, modified nucleic acids, enhanced modified RNA orribonucleic acids of the present invention. As a non-limiting example,in mice bearing a luciferease-expressing tumor, it was determined thatthe lipid-polymer-lipid hybrid nanoparticle significantly suppressedluciferase expression, as compared to a conventional lipoplex (Shi etal, Angew Chem Int Ed. 2011 50:7027-7031).

Peptides and Proteins

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention can be formulated with peptidesand/or proteins in order to increase transfection of cells by themodified nucleic acids, enhanced modified RNA or ribonucleic acids. Inone embodiment, peptides such as, but not limited to, cell penetratingpeptides and proteins and peptides that enable intracellular deliverymay be used to deliver pharmaceutical formulations. A non-limitingexample of a cell penetrating peptide which may be used with thepharmaceutical formulations of the present invention includes acell-penetrating peptide sequence attached to polycations thatfacilitates delivery to the intracellular space, e.g., HIV-derived TATpeptide, penetratins, transportans, or hCT derived cell-penetratingpeptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel,Cell-Penetrating Peptides: Processes and Applications (CRC Press, BocaRaton Fla., 2002); El-Andaloussi et al., Curr. Pharm. Des.11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life Sci.62(16):1839-49 (2005), all of which are incorporated herein byreference). The compositions can also be formulated to include a cellpenetrating agent, e.g., liposomes, which enhance delivery of thecompositions to the intracellular space. Modified nucleic acids,enhanced modified RNA or ribonucleic acids of the invention may becomplexed to peptides and/or proteins such as, but not limited to,peptides and/or proteins from Aileron Therapeutics (Cambridge, Mass.)and Permeon Biologics (Cambridge, Mass.) in order to enableintracellular delivery (Cronican et al., ACS Chem. Biol. 2010 5:747-752;McNaughton et al., Proc. Natl. Acad. Sci. USA 2009 106:6111-6116;Sawyer, Chem Biol Drug Des. 2009 73:3-6; Verdine and Hilinski, MethodsEnzymol. 2012; 503:3-33; all of which are herein incorporated byreference in its entirety).

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but are not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the modified nucleic acids, enhanced modified RNA orribonucleic acids may be introduced.

Formulations of the including peptides or proteins may be used toincrease cell transfection by the polynucleotides, modified nucleicacids, enhanced modified RNA or ribonucleic acids, alter thebiodistribution of the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids (e.g., by targeting specific tissuesor cell types), and/or increase the translation of encoded protein.

Cells

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention can be transfected ex vivo intocells, which are subsequently transplanted into a subject. Asnon-limiting examples, the pharmaceutical compositions may include redblood cells to deliver modified RNA to liver and myeloid cells,virosomes to deliver modified RNA in virus-like particles (VLPs), andelectroporated cells such as, but not limited to, from MAXCYTE®(Gaithersburg, Md.) and from ERYTECH® (Lyon, France) to deliver modifiedRNA. Examples of use of red blood cells, viral particles andelectroporated cells to deliver payloads other than polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acids havebeen documented (Godfrin et al., Expert Opin Biol Ther. 2012 12:127-133;Fang et al., Expert Opin Biol Ther. 2012 12:385-389; Hu et al., ProcNatl Acad Sci USA. 2011 108:10980-10985; Lund et al., Pharm Res. 201027:400-420; Huckriede et al., J Liposome Res. 2007; 17:39-47; Cusi, HumVaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all ofwhich are herein incorporated by reference in its entirety). Themodified RNA may be delivered in synthetic VLPs synthesized by themethods described in International Pub No. WO2011085231 and US Pub No.20110171248, each of which are herein incorporated by reference in theirentireties.

Cell-based formulations of the polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids of the invention may be usedto ensure cell transfection (e.g., in the cellular carrier), alter thebiodistribution of the modified nucleic acids, enhanced modified RNA orribonucleic acids (e.g., by targeting the cell carrier to specifictissues or cell types), and/or increase the translation of encodedprotein.

Introduction into Cells

A variety of methods are known in the art and suitable for introductionof nucleic acid into a cell, including viral and non-viral mediatedtechniques. Examples of typical non-viral mediated techniques include,but are not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microprojectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion.

The technique of sonoporaiton, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to those in theart and are taught for example as it relates to bacteria in US PatentPublication 20100196983 and as it relates to other cell types in, forexample, US Patent Publication 20100009424, each of which areincorporated herein by reference in their entirety.

Electroporation techniques are also well known in the art. In oneembodiment, modified nucleic acids, enhanced modified RNA or ribonucleicacids may be delivered by electroporation as described in Example 11.

Hyaluronidase

The intramuscular or subcutaneous localized injection ofpolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention can include hyaluronidase, whichcatalyzes the hydrolysis of hyaluronan. By catalyzing the hydrolysis ofhyaluronan, a constituent of the interstitial barrier, hyaluronidaselowers the viscosity of hyaluronan, thereby increasing tissuepermeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440; hereinincorporated by reference in its entirety). It is useful to speed theirdispersion and systemic distribution of encoded proteins produced bytransfected cells. Alternatively, the hyaluronidase can be used toincrease the number of cells exposed to a modified nucleic acids,enhanced modified RNA or ribonucleic acids of the invention administeredintramuscularly or subcutaneously.

Nanoparticle Mimics

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention may be encapsulated within and/orabsorbed to a nanoparticle mimic. A nanoparticle mimic can mimic thedelivery function organisms or particles such as, but not limited to,pathogens, viruses, bacteria, fungus, parasites, prions and cells. As anon-limiting example the polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids of the invention may beencapsulated in a non-viron particle which can mimic the deliveryfunction of a virus (see International Pub. No. WO2012006376 hereinincorporated by reference in its entirety).

Nanotubes

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention can be attached or otherwise bound toat least one nanotube such as, but not limited to, rosette nanotubes,rosette nanotubes having twin bases with a linker, carbon nanotubesand/or single-walled carbon nanotubes, The polynucleotides, modifiednucleic acids, enhanced modified RNA or ribonucleic acids may be boundto the nanotubes through forces such as, but not limited to, steric,ionic, covalent and/or other forces.

In one embodiment, the nanotube can release one or more polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acids intocells. The size and/or the surface structure of at least one nanotubemay be altered so as to govern the interaction of the nanotubes withinthe body and/or to attach or bind to the polynucleotides, modifiednucleic acids, enhanced modified RNA or ribonucleic acids disclosedherein. In one embodiment, the building block and/or the functionalgroups attached to the building block of the at least one nanotube maybe altered to adjust the dimensions and/or properties of the nanotube.As a non-limiting example, the length of the nanotubes may be altered tohinder the nanotubes from passing through the holes in the walls ofnormal blood vessels but still small enough to pass through the largerholes in the blood vessels of tumor tissue.

In one embodiment, at least one nanotube may also be coated withdelivery enhancing compounds including polymers, such as, but notlimited to, polyethylene glycol. In another embodiment, at least onenanotube and/or the modified mRNA may be mixed with pharmaceuticallyacceptable excipients and/or delivery vehicles.

In one embodiment, the polynucleotides or modified mRNA are attachedand/or otherwise bound to at least one rosette nanotube. The rosettenanotubes may be formed by a process known in the art and/or by theprocess described in International Publication No. WO2012094304, hereinincorporated by reference in its entirety. At least one modified mRNAmay be attached and/or otherwise bound to at least one rosette nanotubeby a process as described in International Publication No. WO2012094304,herein incorporated by reference in its entirety, where rosettenanotubes or modules forming rosette nanotubes are mixed in aqueousmedia with at least one modified mRNA under conditions which may causeat least one modified mRNA to attach or otherwise bind to the rosettenanotubes.

Conjugates

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention include conjugates, such as amodified nucleic acids, enhanced modified RNA or ribonucleic acidscovalently linked to a carrier or targeting group, or including twoencoding regions that together produce a fusion protein (e.g., bearing atargeting group and therapeutic protein or peptide).

The conjugates of the invention include a naturally occurring substance,such as a protein (e.g., human serum albumin (HSA), low-densitylipoprotein (LDL), high-density lipoprotein (HDL), or globulin); ancarbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin,cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examplesof polyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Representative U.S. patents that teach the preparation of polynucleotideconjugates, particularly to RNA, include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664;6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which isherein incorporated by reference in their entireties.

In one embodiment, the conjugate of the present invention may functionas a carrier for the polynucleotide, modified nucleic acids, enhancedmodified RNA or ribonucleic acids of the present invention. Theconjugate may comprise a cationic polymer such as, but not limited to,polyamine, polylysine, polyalkylenimine, and polyethylenimine which maybe grafted to with poly(ethylene glycol). As a non-limiting example, theconjugate may be similar to the polymeric conjugate and the method ofsynthesizing the polymeric conjugate described in U.S. Pat. No.6,586,524 herein incorporated by reference in its entirety.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell. Targeting groups mayalso include hormones and hormone receptors. They can also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent fucose, or aptamers. The ligand can be, for example, alipopolysaccharide, or an activator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GalNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein.

In one embodiment, pharmaceutical compositions of the present inventionmay include chemical modifications such as, but not limited to,modifications similar to locked nucleic acids.

Representative U.S. patents that teach the preparation of locked nucleicacid (LNA) such as those from Santaris, include, but are not limited to,the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is hereinincorporated by reference in its entirety.

Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found, for example, in Nielsenet al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include modified nucleicacids, enhanced modified RNA or ribonucleic acids with phosphorothioatebackbones and oligonucleosides with other modified backbones, and inparticular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene(methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P(O)₂—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, thepolynucleotides featured herein have morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

Modifications at the 2′ position may also aid in delivery. Preferably,modifications at the 2′ position are not located in a polypeptide-codingsequence, i.e., not in a translatable region. Modifications at the 2′position may be located in a 5′UTR, a 3′UTR and/or a tailing region.Modifications at the 2′ position can include one of the following at the2′ position: H (i.e., 2′-deoxy); F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, the modified nucleicacids, enhanced modified RNA or ribonucleic acids include one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties, or a group for improving thepharmacodynamic properties, and other substituents having similarproperties. In some embodiments, the modification includes a2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl)or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., analkoxy-alkoxy group. Another exemplary modification is2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-β-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below. Othermodifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions, particularly the 3′ position of the sugar onthe 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ positionof 5′ terminal nucleotide. Polynucleotides of the invention may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920 and each of which isherein incorporated by reference.

In still other embodiments, the polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids is covalently conjugated to acell penetrating polypeptide. The cell-penetrating peptide may alsoinclude a signal sequence. The conjugates of the invention can bedesigned to have increased stability; increased cell transfection;and/or altered the biodistribution (e.g., targeted to specific tissuesor cell types).

Self-Assembled Nucleic Acid Nanoparticles

Self-assembled nanoparticles have a well-defined size which may beprecisely controlled as the nucleic acid strands may be easilyreprogrammable. For example, the optimal particle size for acancer-targeting nanodelivery carrier is 20-100 nm as a diameter greaterthan 20 nm avoids renal clearance and enhances delivery to certaintumors through enhanced permeability and retention effect. Usingself-assembled nucleic acid nanoparticles a single uniform population insize and shape having a precisely controlled spatial orientation anddensity of cancer-targeting ligands for enhanced delivery. As anon-limiting example, oligonucleotide nanoparticles were prepared usingprogrammable self-assembly of short DNA fragments and therapeuticsiRNAs. These nanoparticles are molecularly identical with controllableparticle size and target ligand location and density. The DNA fragmentsand siRNAs self-assembled into a one-step reaction to generate DNA/siRNAtetrahedral nanoparticles for targeted in vivo delivery. (Lee et al.,Nature Nanotechnology 2012 7:389-393).

Excipients

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but are notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Various excipients for formulating pharmaceuticalcompositions and techniques for preparing the composition are known inthe art (see Remington: The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference). The use of a conventionalexcipient medium may be contemplated within the scope of the presentdisclosure, except insofar as any conventional excipient medium may beincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition.

In some embodiments, a pharmaceutically acceptable excipient may be atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% pure. In some embodiments, an excipient may be approved for use forhumans and for veterinary use. In some embodiments, an excipient may beapproved by United States Food and Drug Administration. In someembodiments, an excipient may be of pharmaceutical grade. In someembodiments, an excipient may meet the standards of the United StatesPharmacopoeia (USP), the European Pharmacopoeia (EP), the BritishPharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical formulations.The composition may also include excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and/or perfuming agents.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [Span®60], sorbitan tristearate[Span®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER® 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate)(VEEGUM®, and larcharabogalactan); alginates; polyethylene oxide; polyethylene glycol;inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Delivery

The present disclosure encompasses the delivery of polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acids forany of therapeutic, pharmaceutical, diagnostic or imaging by anyappropriate route taking into consideration likely advances in thesciences of drug delivery. Delivery may be naked or formulated.

Naked Delivery

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the present invention may be delivered to a cellnaked. As used herein in, “naked” refers to delivering polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acids freefrom agents which promote transfection. For example, thepolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids delivered to the cell may contain no modifications.The naked polynucleotides, modified nucleic acids, enhanced modified RNAor ribonucleic acids may be delivered to the cell using routes ofadministration known in the art and described herein.

Formulated Delivery

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the present invention may be formulated, using themethods described herein. The formulations may contain polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acids whichmay be modified and/or unmodified. The formulations may further include,but are not limited to, cell penetration agents, a pharmaceuticallyacceptable carrier, a delivery agent, a bioerodible or biocompatiblepolymer, a solvent, and a sustained-release delivery depot. Theformulated polynucleotides, modified nucleic acids or enhanced modifiednucleic acids may be delivered to the cell using routes ofadministration known in the art and described herein.

The compositions may also be formulated for direct delivery to an organor tissue in any of several ways in the art including, but not limitedto, direct soaking or bathing, via a catheter, by gels, powder,ointments, creams, gels, lotions, and/or drops, by using substrates suchas fabric or biodegradable materials coated or impregnated with thecompositions, and the like.

In certain embodiments, the formulations include one or more cellpenetration agents, e.g., transfection agents. In one specificembodiment, a ribonucleic acid is mixed or admixed with a transfectionagent (or mixture thereof) and the resulting mixture is employed totransfect cells. Preferred transfection agents are cationic lipidcompositions, particularly monovalent and polyvalent cationic lipidcompositions, more particularly “LIPOFECTIN,” “LIPOFECTACE,”“LIPOFECTAMINE,” “CELLFECTIN,” DMRIE-C, DMRIE, DOTAP, DOSPA, and DOSPER,and dendrimer compositions, particularly G5-G10 dendrimers, includingdense star dendrimers, PAMAM dendrimers, grafted dendrimers, anddendrimers known as dendrigrafts and “SUPERFECT.” In a second specifictransfection method, a ribonucleic acid is conjugated to a nucleicacid-binding group, for example a polyamine and more particularly aspermine, which is then introduced into the cell or admixed with atransfection agent (or mixture thereof) and the resulting mixture isemployed to transfect cells. In a third specific embodiment, a mixtureof one or more transfection-enhancing peptides, proteins, or proteinfragments, including fusagenic peptides or proteins, transport ortrafficking peptides or proteins, receptor-ligand peptides or proteins,or nuclear localization peptides or proteins and/or their modifiedanalogs (e.g., spermine modified peptides or proteins) or combinationsthereof are mixed with and complexed with a ribonucleic acid to beintroduced into a cell, optionally being admixed with transfection agentand the resulting mixture is employed to transfect cells. Further, acomponent of a transfection agent (e.g., lipids, cationic lipids ordendrimers) is covalently conjugated to selected peptides, proteins, orprotein fragments directly or via a linking or spacer group. Ofparticular interest in this embodiment are peptides or proteins that arefusagenic, membrane-permeabilizing, transport or trafficking, or whichfunction for cell-targeting. The peptide- or protein-transfection agentcomplex is combined with a ribonucleic acid and employed fortransfection.

In certain embodiments, the formulations include a pharmaceuticallyacceptable carrier that causes the effective amount of polynucleotide,modified nucleic acid, or ribonucleic acid to be substantially retainedin a target tissue containing the cell.

Administration

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the present invention may be administered by anyroute which results in a therapeutically effective outcome. Theseinclude, but are not limited to enteral, gastroenteral, epidural, oral,transdermal, epidural (peridural), intracerebral (into the cerebrum),intracerebroventricular (into the cerebral ventricles), epicutaneous(application onto the skin), intradermal, (into the skin itself),subcutaneous (under the skin), nasal administration (through the nose),intravenous (into a vein), intraarterial (into an artery), intramuscular(into a muscle), intracardiac (into the heart), intraosseous infusion(into the bone marrow), intrathecal (into the spinal canal),intraperitoneal, (infusion or injection into the peritoneum),intravesical infusion, intravitreal, (through the eye), intracavernousinjection, (into the base of the penis), intravaginal administration,intrauterine, extra-amniotic administration, transdermal (diffusionthrough the intact skin for systemic distribution), transmucosal(diffusion through a mucous membrane), insufflation (snorting),sublingual, sublabial, enema, eye drops (onto the conjunctiva), or inear drops.

In one embodiment, provided are compositions for generation of an invivo depot containing a polynucleotide, modified nucleic acid orengineered ribonucleotide. For example, the composition contains abioerodible, biocompatible polymer, a solvent present in an amounteffective to plasticize the polymer and form a gel therewith, and apolynucleotide, modified nucleic acid or engineered ribonucleic acid. Incertain embodiments the composition also includes a cell penetrationagent as described herein. In other embodiments, the composition alsocontains a thixotropic amount of a thixotropic agent mixable with thepolymer so as to be effective to form a thixotropic composition. Furthercompositions include a stabilizing agent, a bulking agent, a chelatingagent, or a buffering agent.

In other embodiments, provided are sustained-release delivery depots,such as for administration of a polynucleotide, modified nucleic acid,or engineered ribonucleic acid an environment (meaning an organ ortissue site) in a patient. Such depots generally contain an engineeredribonucleic acid and a flexible chain polymer where both the engineeredribonucleic acid and the flexible chain polymer are entrapped within aporous matrix of a crosslinked matrix protein. Usually, the pore size isless than 1 mm, such as 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm,300 nm, 200 nm, 100 nm, or less than 100 nm. Usually the flexible chainpolymer is hydrophilic. Usually the flexible chain polymer has amolecular weight of at least 50 kDa, such as 75 kDa, 100 kDa, 150 kDa,200 kDa, 250 kDa, 300 kDa, 400 kDa, 500 kDa, or greater than 500 kDa.Usually the flexible chain polymer has a persistence length of less than10%, such as 9, 8, 7, 6, 5, 4, 3, 2, 1 or less than 1% of thepersistence length of the matrix protein. Usually the flexible chainpolymer has a charge similar to that of the matrix protein. In someembodiments, the flexible chain polymer alters the effective pore sizeof a matrix of crosslinked matrix protein to a size capable ofsustaining the diffusion of the engineered ribonucleic acid from thematrix into a surrounding tissue comprising a cell into which thepolynucleotide, modified nucleic acid, engineered ribonucleic acid iscapable of entering.

In specific embodiments, compositions may be administered in a way whichallows them cross the blood-brain barrier, vascular barrier, or otherepithelial barrier. Non-limiting routes of administration for thepolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids of the present invention are described below.

The present invention provides methods comprising administeringpolynucleotides, modified mRNAs and their encoded proteins or complexesin accordance with the invention to a subject in need thereof. Nucleicacids, proteins or complexes, or pharmaceutical, imaging, diagnostic, orprophylactic compositions thereof, may be administered to a subjectusing any amount and any route of administration effective forpreventing, treating, diagnosing, or imaging a disease, disorder, and/orcondition (e.g., a disease, disorder, and/or condition relating toworking memory deficits). The exact amount required will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the disease, the particular composition,its mode of administration, its mode of activity, and the like.Compositions in accordance with the invention are typically formulatedin dosage unit form for ease of administration and uniformity of dosage.It will be understood, however, that the total daily usage of thecompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective, prophylactically effective, or appropriateimaging dose level for any particular patient will depend upon a varietyof factors including the disorder being treated and the severity of thedisorder; the activity of the specific compound employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration, andrate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

Parenteral and Injectable Administration

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as CREMOPHOR®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Oral Administration

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g. starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g. agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g. paraffin), absorptionaccelerators (e.g. quaternary ammonium compounds), wetting agents (e.g.cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin andbentonite clay), and lubricants (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate), andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may comprise buffering agents.

Topical or Transdermal Administration

As described herein, compositions containing the polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acids ofthe invention may be formulated for administration topically. The skinmay be an ideal target site for delivery as it is readily accessible.Gene expression may be restricted not only to the skin, potentiallyavoiding nonspecific toxicity, but also to specific layers and celltypes within the skin.

The site of cutaneous expression of the delivered compositions willdepend on the route of nucleic acid delivery. Three routes are commonlyconsidered to deliver polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids to the skin: (i) topical application(e.g. for local/regional treatment); (ii) intradermal injection (e.g.for local/regional treatment); and (iii) systemic delivery (e.g. fortreatment of dermatologic diseases that affect both cutaneous andextracutaneous regions). Polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids can be delivered to the skinby several different approaches known in the art. Most topical deliveryapproaches have been shown to work for delivery of DNA, such as but notlimited to, topical application of non-cationic liposome-DNA complex,cationic liposome-DNA complex, particle-mediated (gene gun),puncture-mediated gene transfections, and viral delivery approaches.After delivery of the nucleic acid, gene products have been detected ina number of different skin cell types, including, but not limited to,basal keratinocytes, sebaceous gland cells, dermal fibroblasts anddermal macrophages.

In one embodiment, the invention provides for a variety of dressings(e.g., wound dressings) or bandages (e.g., adhesive bandages) forconveniently and/or effectively carrying out methods of the presentinvention. Typically dressing or bandages may comprise sufficientamounts of pharmaceutical compositions and/or polynucleotides, modifiednucleic acids, enhanced modified RNA or ribonucleic acids describedherein to allow a user to perform multiple treatments of a subject(s).

In one embodiment, the invention provides for the polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acidscompositions to be delivered in more than one injection.

In one embodiment, before topical and/or transdermal administration atleast one area of tissue, such as skin, may be subjected to a deviceand/or solution which may increase permeability. In one embodiment, thetissue may be subjected to an abrasion device to increase thepermeability of the skin (see U.S. Patent Publication No. 20080275468,herein incorporated by reference in its entirety). In anotherembodiment, the tissue may be subjected to an ultrasound enhancementdevice. An ultrasound enhancement device may include, but is not limitedto, the devices described in U.S. Publication No. 20040236268 and U.S.Pat. Nos. 6,491,657 and 6,234,990; each of which are herein incorporatedby reference in their entireties. Methods of enhancing the permeabilityof tissue are described in U.S. Publication Nos. 20040171980 and20040236268 and U.S. Pat. No. 6,190,315; each of which are hereinincorporated by reference in their entireties.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein.The permeability of skin may be measured by methods known in the artand/or described in U.S. Pat. No. 6,190,315, herein incorporated byreference in its entirety. As a non-limiting example, a modified mRNAformulation may be delivered by the drug delivery methods described inU.S. Pat. No. 6,190,315, herein incorporated by reference in itsentirety.

In another non-limiting example tissue may be treated with a eutecticmixture of local anesthetics (EMLA) cream before, during and/or afterthe tissue may be subjected to a device which may increase permeability.Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated byreference in its entirety) showed that using the EMLA cream incombination with a low energy, an onset of superficial cutaneousanalgesia was seen as fast as 5 minutes after a pretreatment with a lowenergy ultrasound.

In one embodiment, enhancers may be applied to the tissue before,during, and/or after the tissue has been treated to increasepermeability. Enhancers include, but are not limited to, transportenhancers, physical enhancers, and cavitation enhancers. Non-limitingexamples of enhancers are described in U.S. Pat. No. 6,190,315, hereinincorporated by reference in its entirety.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein,which may further contain a substance that invokes an immune response.In another non-limiting example, a formulation containing a substance toinvoke an immune response may be delivered by the methods described inU.S. Publication Nos. 20040171980 and 20040236268; each of which areherein incorporated by reference in their entireties.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present inventioncontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.

Topically-administrable formulations may, for example, comprise fromabout 0.1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Depot Administration

As described herein, in some embodiments, the composition is formulatedin depots for extended release. Generally, a specific organ or tissue (a“target tissue”) is targeted for administration.

In some aspects of the invention, the nucleic acids (particularlyribonucleic acids encoding polypeptides) are spatially retained withinor proximal to a target tissue. Provided are method of providing acomposition to a target tissue of a mammalian subject by contacting thetarget tissue (which contains one or more target cells) with thecomposition under conditions such that the composition, in particularthe nucleic acid component(s) of the composition, is substantiallyretained in the target tissue, meaning that at least 10, 20, 30, 40, 50,60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than99.99% of the composition is retained in the target tissue.Advantageously, retention is determined by measuring the amount of thenucleic acid present in the composition that enters one or more targetcells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85,90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of thenucleic acids administered to the subject are present intracellularly ata period of time following administration. For example, intramuscularinjection to a mammalian subject is performed using an aqueouscomposition containing a ribonucleic acid and a transfection reagent,and retention of the composition is determined by measuring the amountof the ribonucleic acid present in the muscle cells.

Aspects of the invention are directed to methods of providing acomposition to a target tissue of a mammalian subject, by contacting thetarget tissue (containing one or more target cells) with the compositionunder conditions such that the composition is substantially retained inthe target tissue. In another embodiment, a polynucleotide, ribonucleicacid engineered to avoid an innate immune response of a cell into whichthe ribonucleic acid enters, where the ribonucleic acid contains anucleotide sequence encoding a polypeptide of interest, under conditionssuch that the polypeptide of interest is produced in at least one targetcell. The compositions generally contain a cell penetration agent,although “naked” nucleic acid (such as nucleic acids without a cellpenetration agent or other agent) is also contemplated, and apharmaceutically acceptable carrier.

In some circumstances, the amount of a protein produced by cells in atissue is desirably increased. Preferably, this increase in proteinproduction is spatially restricted to cells within the target tissue.Thus, provided are methods of increasing production of a protein ofinterest in a tissue of a mammalian subject. A composition is providedthat contains a ribonucleic acid that is engineered to avoid an innateimmune response of a cell into which the ribonucleic acid enters andencodes the polypeptide of interest and the composition is characterizedin that a unit quantity of composition has been determined to producethe polypeptide of interest in a substantial percentage of cellscontained within a predetermined volume of the target tissue.

In some embodiments, the composition includes a plurality of differentribonucleic acids, where one or more than one of the ribonucleic acidsis engineered to avoid an innate immune response of a cell into whichthe ribonucleic acid enters, and where one or more than one of theribonucleic acids encodes a polypeptide of interest. Optionally, thecomposition also contains a cell penetration agent to assist in theintracellular delivery of the ribonucleic acid. A determination is madeof the dose of the composition required to produce the polypeptide ofinterest in a substantial percentage of cells contained within thepredetermined volume of the target tissue (generally, without inducingsignificant production of the polypeptide of interest in tissue adjacentto the predetermined volume, or distally to the target tissue).Subsequent to this determination, the determined dose is introduceddirectly into the tissue of the mammalian subject.

In one embodiment, the invention provides for the polynucleotides,modified nucleic acids, enhanced modified RNA or ribonucleic acids to bedelivered in more than one injection or by split dose injections.

In one embodiment, the invention may be retained near target tissueusing a small disposable drug reservoir or patch pump. Non-limitingexamples of patch pumps include those manufactured and/or sold by BD®,(Franklin Lakes, N.J.), Insulet Corporation (Bedford, Mass.), SteadyMedTherapeutics (San Francisco, Calif.), Medtronic (Minneapolis, Minn.),UniLife (York, Pa.), Valeritas (Bridgewater, N.J.), and SpringLeafTherapeutics (Boston, Mass.).

Pulmonary Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions aresuitably in the form of dry powders for administration using a devicecomprising a dry powder reservoir to which a stream of propellant may bedirected to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further comprise additional ingredients such as a liquidnon-ionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles comprising theactive ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Intranasal, Nasal and Buccal Administration

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein

Ophthalmic Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis invention.

Payload Administration: Detectable Agents and Therapeutic Agents

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids described herein can be used in a number of differentscenarios in which delivery of a substance (the “payload”) to abiological target is desired, for example delivery of detectablesubstances for detection of the target, or delivery of a therapeuticagent. Detection methods can include, but are not limited to, bothimaging in vitro and in vivo imaging methods, e.g.,immunohistochemistry, bioluminescence imaging (BLI), Magnetic ResonanceImaging (MRI), positron emission tomography (PET), electron microscopy,X-ray computed tomography, Raman imaging, optical coherence tomography,absorption imaging, thermal imaging, fluorescence reflectance imaging,fluorescence microscopy, fluorescence molecular tomographic imaging,nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging,photoacoustic imaging, lab assays, or in any situation wheretagging/staining/imaging is required.

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids can be designed to include both a linker and a payloadin any useful orientation. For example, a linker having two ends is usedto attach one end to the payload and the other end to the nucleobase,such as at the C-7 or C-8 positions of the deaza-adenosine ordeaza-guanosine or to the N-3 or C-5 positions of cytosine or uracil.The polynucleotide of the invention can include more than one payload(e.g., a label and a transcription inhibitor), as well as a cleavablelinker.

In one embodiment, the modified nucleotide is a modified7-deaza-adenosine triphosphate, where one end of a cleavable linker isattached to the C7 position of 7-deaza-adenine, the other end of thelinker is attached to an inhibitor (e.g., to the C5 position of thenucleobase on a cytidine), and a label (e.g., Cy5) is attached to thecenter of the linker (see, e.g., compound 1 of A*pCp C5 Parg Capless inFIG. 5 and columns 9 and 10 of U.S. Pat. No. 7,994,304, incorporatedherein by reference). Upon incorporation of the modified7-deaza-adenosine triphosphate to an encoding region, the resultingpolynucleotide having a cleavable linker attached to a label and aninhibitor (e.g., a polymerase inhibitor). Upon cleavage of the linker(e.g., with reductive conditions to reduce a linker having a cleavabledisulfide moiety), the label and inhibitor are released. Additionallinkers and payloads (e.g., therapeutic agents, detectable labels, andcell penetrating payloads) are described herein.

For example, the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids described herein can be used inreprogramming induced pluripotent stem cells (iPS cells), which candirectly track cells that are transfected compared to total cells in thecluster. In another example, a drug that may be attached to the modifiednucleic acids, enhanced modified RNA or ribonucleic acids via a linkerand may be fluorescently labeled can be used to track the drug in vivo,e.g. intracellularly. Other examples include, but are not limited to,the use of polynucleotides, modified nucleic acids, enhanced modifiedRNA or ribonucleic acids in reversible drug delivery into cells.

The polynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids described herein can be used in intracellulartargeting of a payload, e.g., detectable or therapeutic agent, tospecific organelle. Exemplary intracellular targets can include, but arenot limited to, the nuclear localization for advanced mRNA processing,or a nuclear localization sequence (NLS) linked to the mRNA containingan inhibitor.

In addition, the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids described herein can be used todeliver therapeutic agents to cells or tissues, e.g., in living animals.For example, the polynucleotides, modified nucleic acids, enhancedmodified RNA or ribonucleic acids described herein can be used todeliver highly polar chemotherapeutics agents to kill cancer cells. Thepolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids attached to the therapeutic agent through a linker canfacilitate member permeation allowing the therapeutic agent to travelinto a cell to reach an intracellular target.

In another example, the polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids can be attached to thepolynucleotides, modified nucleic acids, enhanced modified RNA orribonucleic acids a viral inhibitory peptide (VIP) through a cleavablelinker. The cleavable linker can release the VIP and dye into the cell.In another example, the polynucleotides, modified nucleic acids,enhanced modified RNA or ribonucleic acids can be attached through thelinker to an ADP-ribosylate, which is responsible for the actions ofsome bacterial toxins, such as cholera toxin, diphtheria toxin, andpertussis toxin. These toxin proteins are ADP-ribosyltransferases thatmodify target proteins in human cells. For example, cholera toxinADP-ribosylates G proteins modifies human cells by causing massive fluidsecretion from the lining of the small intestine, which results inlife-threatening diarrhea.

In some embodiments, the payload may be a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that may bedetrimental to cells. Examples include, but are not limited to, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, teniposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein inits entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,5,585,499, and 5,846,545, all of which are incorporated herein byreference), and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

In some embodiments, the payload may be a detectable agent, such asvarious organic small molecules, inorganic compounds, nanoparticles,enzymes or enzyme substrates, fluorescent materials, luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin, and aequorin), chemiluminescent materials, radioactivematerials (e.g., ¹⁸F, ⁶⁷Ga, ^(81m)KR, ⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl,¹²⁵I, ³⁵S, ¹⁴C, ³H, or ^(99m)Tc (e.g., as pertechnetate(technetate(VII), TcO₄ ⁻)), and contrast agents (e.g., gold (e.g., goldnanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g.,superparamagnetic iron oxide (SPIO), monocrystalline iron oxidenanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide(USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinatedcontrast media (iohexyl), microbubbles, or perfluorocarbons). Suchoptically-detectable labels include for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (Cibacron™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red),N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolicacid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine

In some embodiments, the detectable agent may be a non-detectablepre-cursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Invitro assays in which the enzyme labeled compositions can be usedinclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), immunoprecipitation assays, immunofluorescence, enzymeimmunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.Combination

The modified nucleic acids, enhanced modified RNA or ribonucleic acidsmay be used in combination with one or more other therapeutic,prophylactic, diagnostic, or imaging agents. By “in combination with,”it is not intended to imply that the agents must be administered at thesame time and/or formulated for delivery together, although thesemethods of delivery are within the scope of the present disclosure.Compositions can be administered concurrently with, prior to, orsubsequent to, one or more other desired therapeutics or medicalprocedures. In general, each agent will be administered at a dose and/oron a time schedule determined for that agent. In some embodiments, thepresent disclosure encompasses the delivery of pharmaceutical,prophylactic, diagnostic, or imaging compositions in combination withagents that may improve their bioavailability, reduce and/or modifytheir metabolism, inhibit their excretion, and/or modify theirdistribution within the body. As a non-limiting example, the modifiednucleic acids, enhanced modified RNA or ribonucleic acids may be used incombination with a pharmaceutical agent for the treatment of cancer orto control hyperproliferative cells. In U.S. Pat. No. 7,964,571, hereinincorporated by reference in its entirety, a combination therapy for thetreatment of solid primary or metastasized tumor is described using apharmaceutical composition including a DNA plasmid encoding forinterleukin-12 with a lipopolymer and also administering at least oneanticancer agent or chemotherapeutic. Further, the modified nucleicacids, enhanced modified RNA or ribonucleic acids of the presentinvention that encodes anti-proliferative molecules may be in apharmaceutical composition with a lipopolymer (see e.g., U.S. Pub. No.20110218231, herein incorporated by reference in its entirety, claiminga pharmaceutical composition comprising a DNA plasmid encoding ananti-proliferative molecule and a lipopolymer) which may be administeredwith at least one chemotherapeutic or anticancer agent.

Payload Administration: Cell Penetrating Payload

In some embodiments, the polynucleotides, modified nucleotides andmodified nucleic acid molecules, which are incorporated into a nucleicacid, e.g., RNA or mRNA, can also include a payload that can be a cellpenetrating moiety or agent that enhances intracellular delivery of thecompositions. For example, the compositions can include, but are notlimited to, a cell-penetrating peptide sequence that facilitatesdelivery to the intracellular space, e.g., HIV-derived TAT peptide,penetratins, transportans, or hCT derived cell-penetrating peptides,see, e.g., Caron et al., (2001) Mol. Ther. 3(3):310-8; Langel,Cell-Penetrating Peptides: Processes and Applications (CRC Press, BocaRaton Fla. 2002); El-Andaloussi et al., (2005) Curr Pharm Des.11(28):3597-611; and Deshayes et al., (2005) Cell Mol Life Sci.62(16):1839-49; all of which are incorporated herein by reference. Thecompositions can also be formulated to include a cell penetrating agent,e.g., liposomes, which enhance delivery of the compositions to theintracellular space.

Payload Administration: Biological Target

The modified nucleotides and modified nucleic acid molecules describedherein, which are incorporated into a nucleic acid, e.g., RNA or mRNA,can be used to deliver a payload to any biological target for which aspecific ligand exists or can be generated. The ligand can bind to thebiological target either covalently or non-covalently.

Examples of biological targets include, but are not limited to,biopolymers, e.g., antibodies, nucleic acids such as RNA and DNA,proteins, enzymes; examples of proteins include, but are not limited to,enzymes, receptors, and ion channels. In some embodiments the target maybe a tissue- or a cell-type specific marker, e.g., a protein that isexpressed specifically on a selected tissue or cell type. In someembodiments, the target may be a receptor, such as, but not limited to,plasma membrane receptors and nuclear receptors; more specific examplesinclude, but are not limited to, G-protein-coupled receptors, cell poreproteins, transporter proteins, surface-expressed antibodies, HLAproteins, MHC proteins and growth factor receptors.

Dosing

The present invention provides methods comprising administering modifiedmRNAs and their encoded proteins or complexes in accordance with theinvention to a subject in need thereof. Nucleic acids, proteins orcomplexes, or pharmaceutical, imaging, diagnostic, or prophylacticcompositions thereof, may be administered to a subject using any amountand any route of administration effective for preventing, treating,diagnosing, or imaging a disease, disorder, and/or condition (e.g., adisease, disorder, and/or condition relating to working memorydeficits). The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Compositions inaccordance with the invention are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention may be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect. The desired dosage may be deliveredthree times a day, two times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

According to the present invention, it has been discovered thatadministration of modified nucleic acids, enhanced modified RNA orribonucleic acids in split-dose regimens produce higher levels ofproteins in mammalian subjects. As used herein, a “split dose” is thedivision of single unit dose or total daily dose into two or more doses,e.g, two or more administrations of the single unit dose. As usedherein, a “single unit dose” is a dose of any therapeutic administeredin one dose/at one time/single route/single point of contact, i.e.,single administration event. As used herein, a “total daily dose” is anamount given or prescribed in 24 hr period. It may be administered as asingle unit dose. In one embodiment, the modified nucleic acids,enhanced modified RNA or ribonucleic acids of the present invention areadministered to a subject in split doses. The modified nucleic acids,enhanced modified RNA or ribonucleic acids may be formulated in bufferonly or in a formulation described herein.

Dosage Forms

A pharmaceutical composition described herein can be formulated into adosage form described herein, such as a topical, intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intracardiac, intraperitoneal,subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art including, but not limited to, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. In certainembodiments for parenteral administration, compositions may be mixedwith solubilizing agents such as CREMOPHOR®, alcohols, oils, modifiedoils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known art andmay include suitable dispersing agents, wetting agents, and/orsuspending agents. Sterile injectable preparations may be sterileinjectable solutions, suspensions, and/or emulsions in nontoxicparenterally acceptable diluents and/or solvents, for example, asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed include, but are not limited to, are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution.Sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil can be employedincluding synthetic mono- or diglycerides. Fatty acids such as oleicacid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it may bedesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of modified mRNA thendepends upon its rate of dissolution which, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered modified mRNA may be accomplished bydissolving or suspending the modified mRNA in an oil vehicle. Injectabledepot forms are made by forming microencapsule matrices of the modifiedmRNA in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of modified mRNA to polymer and the nature ofthe particular polymer employed, the rate of modified mRNA release canbe controlled. Examples of other biodegradable polymers include, but arenot limited to, poly(orthoesters) and poly(anhydrides). Depot injectableformulations may be prepared by entrapping the modified mRNA inliposomes or microemulsions which are compatible with body tissues.

Pulmonary

Formulations described herein as being useful for pulmonary delivery mayalso be use for intranasal delivery of a pharmaceutical composition.Another formulation suitable for intranasal administration may be acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 μm to 500 μm. Such a formulation may beadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, contain about 0.1% to 20% (w/w) active ingredient, where thebalance may comprise an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods of the present invention. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments.

In one aspect, the present invention provides kits for proteinproduction, comprising a first modified nucleic acids, enhanced modifiedRNA or ribonucleic acids comprising a translatable region. The kit mayfurther comprise packaging and instructions and/or a delivery agent toform a formulation composition. The delivery agent may comprise asaline, a buffered solution, a lipidoid or any delivery agent disclosedherein.

In one embodiment, the buffer solution may include sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution may include, but is not limited to, saline, saline with2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium. In a further embodiment, the buffersolutions may be precipitated or it may be lyophilized. The amount ofeach component may be varied to enable consistent, reproducible higherconcentration saline or simple buffer formulations. The components mayalso be varied in order to increase the stability of modified RNA in thebuffer solution over a period of time and/or under a variety ofconditions.

In one aspect, the present invention provides kits for proteinproduction, comprising: a modified nucleic acids, enhanced modified RNAor ribonucleic acids comprising a translatable region, provided in anamount effective to produce a desired amount of a protein encoded by thetranslatable region when introduced into a target cell; a secondmodified nucleic acids, enhanced modified RNA or ribonucleic acidscomprising an inhibitory nucleic acid, provided in an amount effectiveto substantially inhibit the innate immune response of the cell; andpackaging and instructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a modified nucleic acids, enhanced modified RNAor ribonucleic acids comprising a translatable region, wherein thenucleic acid exhibits reduced degradation by a cellular nuclease, andpackaging and instructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a modified nucleic acids, enhanced modified RNAor ribonucleic acids comprising a translatable region, wherein thenucleic acid exhibits reduced degradation by a cellular nuclease, and amammalian cell suitable for translation of the translatable region ofthe first nucleic acid

Devices

The present invention provides for devices which may incorporatemodified nucleic acids, enhanced modified RNA or ribonucleic acids thatencode polypeptides of interest. These devices contain in a stableformulation the reagents to synthesize a nucleic acid in a formulationavailable to be immediately delivered to a subject in need thereof, suchas a human patient. Non-limiting examples of such a polypeptide ofinterest include a growth factor and/or angiogenesis stimulator forwound healing, a peptide antibiotic to facilitate infection control, andan antigen to rapidly stimulate an immune response to a newly identifiedvirus.

In some embodiments the device is self-contained, and is optionallycapable of wireless remote access to obtain instructions for synthesisand/or analysis of the generated modified nucleic acids, enhancedmodified RNA or ribonucleic acids. The device is capable of mobilesynthesis of at least one modified nucleic acids, enhanced modified RNAor ribonucleic acids and preferably an unlimited number of differentmodified nucleic acids, enhanced modified RNA or ribonucleic acids. Incertain embodiments, the device is capable of being transported by oneor a small number of individuals. In other embodiments, the device isscaled to fit on a benchtop or desk. In other embodiments, the device isscaled to fit into a suitcase, backpack or similarly sized object. Inanother embodiment, the device may be a point of care or handhelddevice. In further embodiments, the device is scaled to fit into avehicle, such as a car, truck or ambulance, or a military vehicle suchas a tank or personnel carrier. The information necessary to generate aribonucleic acid encoding polypeptide of interest is present within acomputer readable medium present in the device.

In one embodiment, a device may be used to assess levels of a proteinwhich has been administered in the form of a modified nucleic acids,enhanced modified RNA or ribonucleic acids. The device may comprise ablood, urine or other biofluidic test.

In some embodiments, the device is capable of communication (e.g.,wireless communication) with a database of nucleic acid and polypeptidesequences. The device contains at least one sample block for insertionof one or more sample vessels. Such sample vessels are capable ofaccepting in liquid or other form any number of materials such astemplate DNA, nucleotides, enzymes, buffers, and other reagents. Thesample vessels are also capable of being heated and cooled by contactwith the sample block. The sample block is generally in communicationwith a device base with one or more electronic control units for the atleast one sample block. The sample block preferably contains a heatingmodule, such heating molecule capable of heating and/or cooling thesample vessels and contents thereof to temperatures between about −20 Cand above +100 C. The device base is in communication with a voltagesupply such as a battery or external voltage supply. The device alsocontains means for storing and distributing the materials for RNAsynthesis.

Optionally, the sample block contains a module for separating thesynthesized nucleic acids. Alternatively, the device contains aseparation module operably linked to the sample block. Preferably thedevice contains a means for analysis of the synthesized nucleic acid.Such analysis includes sequence identity (demonstrated such as byhybridization), absence of non-desired sequences, measurement ofintegrity of synthesized mRNA (such has by microfluidic viscometrycombined with spectrophotometry), and concentration and/or potency ofmodified nucleic acids, enhanced modified RNA or ribonucleic acids (suchas by spectrophotometry).

In certain embodiments, the device is combined with a means fordetection of pathogens present in a biological material obtained from asubject, e.g., the IBIS PLEX-ID system (Abbott, Abbott Park, Ill.) formicrobial identification.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid compositions to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable.

Alternatively or additionally, conventional syringes may be used in theclassical mantoux method of intradermal administration.

In some embodiments, the device may be a pump or comprise a catheter foradministration of compounds or compositions of the invention across theblood brain barrier. Such devices include but are not limited to apressurized olfactory delivery device, iontophoresis devices,multi-layered microfluidic devices, and the like. Such devices may beportable or stationary. They may be implantable or externally tetheredto the body or combinations thereof.

Devices for administration may be employed to deliver the modifiednucleic acids, enhanced modified RNA or ribonucleic acids of the presentinvention according to single, multi- or split-dosing regimens taughtherein. Such devices are described below.

Method and devices known in the art for multi-administration to cells,organs and tissues are contemplated for use in conjunction with themethods and compositions disclosed herein as embodiments of the presentinvention. These include, for example, those methods and devices havingmultiple needles, hybrid devices employing for example lumens orcatheters as well as devices utilizing heat, electric current orradiation driven mechanisms.

According to the present invention, these multi-administration devicesmay be utilized to deliver the single, multi- or split dosescontemplated herein.

A method for delivering therapeutic agents to a solid tissue has beendescribed by Bahrami et al. and is taught for example in US PatentPublication 20110230839, the contents of which are incorporated hereinby reference in their entirety. According to Bahrami, an array ofneedles is incorporated into a device which delivers a substantiallyequal amount of fluid at any location in said solid tissue along eachneedle's length.

A device for delivery of biological material across the biologicaltissue has been described by Kodgule et al. and is taught for example inUS Patent Publication 20110172610, the contents of which areincorporated herein by reference in their entirety. According toKodgule, multiple hollow micro-needles made of one or more metals andhaving outer diameters from about 200 microns to about 350 microns andlengths of at least 100 microns are incorporated into the device whichdelivers peptides, proteins, carbohydrates, nucleic acid molecules,lipids and other pharmaceutically active ingredients or combinationsthereof.

A delivery probe for delivering a therapeutic agent to a tissue has beendescribed by Gunday et al. and is taught for example in US PatentPublication 20110270184, the contents of which are incorporated hereinby reference in their entirety. According to Gunday, multiple needlesare incorporated into the device which moves the attached capsulesbetween an activated position and an inactivated position to force theagent out of the capsules through the needles.

A multiple-injection medical apparatus has been described by Assaf andis taught for example in US Patent Publication 20110218497, the contentsof which are incorporated herein by reference in their entirety.According to Assaf, multiple needles are incorporated into the devicewhich has a chamber connected to one or more of said needles and a meansfor continuously refilling the chamber with the medical fluid after eachinjection.

In one embodiment, the modified nucleic acids, enhanced modified RNA orribonucleic acids are administered subcutaneously or intramuscularly viaat least 3 needles to three different, optionally adjacent, sitessimultaneously, or within a 60 minutes period (e.g., administration to4, 5, 6, 7, 8, 9, or 10 sites simultaneously or within a 60 minuteperiod). The split doses can be administered simultaneously to adjacenttissue using the devices described in U.S. Patent Publication Nos.20110230839 and 20110218497, each of which is incorporated herein byreference.

An at least partially implantable system for injecting a substance intoa patient's body, in particular a penis erection stimulation system hasbeen described by Forsell and is taught for example in US PatentPublication 20110196198, the contents of which are incorporated hereinby reference in their entirety. According to Forsell, multiple needlesare incorporated into the device which is implanted along with one ormore housings adjacent the patient's left and right corpora cavernosa. Areservoir and a pump are also implanted to supply drugs through theneedles.

A method for the transdermal delivery of a therapeutic effective amountof iron has been described by Berenson and is taught for example in USPatent Publication 20100130910, the contents of which are incorporatedherein by reference in their entirety. According to Berenson, multipleneedles may be used to create multiple micro channels in stratum corneumto enhance transdermal delivery of the ionic iron on an iontophoreticpatch.

A method for delivery of biological material across the biologicaltissue has been described by Kodgule et al and is taught for example inUS Patent Publication 20110196308, the contents of which areincorporated herein by reference in their entirety. According toKodgule, multiple biodegradable microneedles containing a therapeuticactive ingredient are incorporated in a device which delivers proteins,carbohydrates, nucleic acid molecules, lipids and other pharmaceuticallyactive ingredients or combinations thereof.

A transdermal patch comprising a botulinum toxin composition has beendescribed by Donovan and is taught for example in US Patent Publication20080220020, the contents of which are incorporated herein by referencein their entirety. According to Donovan, multiple needles areincorporated into the patch which delivers botulinum toxin under stratumcorneum through said needles which project through the stratum corneumof the skin without rupturing a blood vessel.

A small, disposable drug reservoir, or patch pump, which can holdapproximately 0.2 to 15 mL of liquid formulations can be placed on theskin and deliver the formulation continuously subcutaneously using asmall bore needed (e.g., 26 to 34 gauge). As non-limiting examples, thepatch pump may be 50 mm by 76 mm by 20 mm spring loaded having a 30 to34 gauge needle (BD™ Microinfuser, Franklin Lakes N.J.), 41 mm by 62 mmby 17 mm with a 2 mL reservoir used for drug delivery such as insulin(OMNIPOD®, Insulet Corporation Bedford, Mass.), or 43-60 mm diameter, 10mm thick with a 0.5 to 10 mL reservoir (PATCHPUMP®, SteadyMedTherapeutics, San Francisco, Calif.). Further, the patch pump may bebattery powered and/or rechargeable.

A cryoprobe for administration of an active agent to a location ofcryogenic treatment has been described by Toubia and is taught forexample in US Patent Publication 20080140061, the contents of which areincorporated herein by reference in their entirety. According to Toubia,multiple needles are incorporated into the probe which receives theactive agent into a chamber and administers the agent to the tissue.

A method for treating or preventing inflammation or promoting healthyjoints has been described by Stock et al and is taught for example in USPatent Publication 20090155186, the contents of which are incorporatedherein by reference in their entirety. According to Stock, multipleneedles are incorporated in a device which administers compositionscontaining signal transduction modulator compounds.

A multi-site injection system has been described by Kimmell et al. andis taught for example in US Patent Publication 20100256594, the contentsof which are incorporated herein by reference in their entirety.According to Kimmell, multiple needles are incorporated into a devicewhich delivers a medication into a stratum corneum through the needles.

A method for delivering interferons to the intradermal compartment hasbeen described by Dekker et al. and is taught for example in US PatentPublication 20050181033, the contents of which are incorporated hereinby reference in their entirety. According to Dekker, multiple needleshaving an outlet with an exposed height between 0 and 1 mm areincorporated into a device which improves pharmacokinetics andbioavailability by delivering the substance at a depth between 0.3 mmand 2 mm.

A method for delivering genes, enzymes and biological agents to tissuecells has described by Desai and is taught for example in US PatentPublication 20030073908, the contents of which are incorporated hereinby reference in their entirety. According to Desai, multiple needles areincorporated into a device which is inserted into a body and delivers amedication fluid through said needles.

A method for treating cardiac arrhythmias with fibroblast cells has beendescribed by Lee et al and is taught for example in US PatentPublication 20040005295, the contents of which are incorporated hereinby reference in their entirety. According to Lee, multiple needles areincorporated into the device which delivers fibroblast cells into thelocal region of the tissue.

A method using a magnetically controlled pump for treating a brain tumorhas been described by Shachar et al. and is taught for example in U.S.Pat. No. 7,799,012 (method) and U.S. Pat. No. 7,799,016 (device), thecontents of which are incorporated herein by reference in theirentirety. According Shachar, multiple needles were incorporated into thepump which pushes a medicating agent through the needles at a controlledrate.

Methods of treating functional disorders of the bladder in mammalianfemales have been described by Versi et al. and are taught for examplein U.S. Pat. No. 8,029,496, the contents of which are incorporatedherein by reference in their entirety. According to Versi, an array ofmicro-needles is incorporated into a device which delivers a therapeuticagent through the needles directly into the trigone of the bladder.

A micro-needle transdermal transport device has been described by Angelet al and is taught for example in U.S. Pat. No. 7,364,568, the contentsof which are incorporated herein by reference in their entirety.According to Angel, multiple needles are incorporated into the devicewhich transports a substance into a body surface through the needleswhich are inserted into the surface from different directions. Themicro-needle transdermal transport device may be a solid micro-needlesystem or a hollow micro-needle system. As a non-limiting example, thesolid micro-needle system may have up to a 0.5 mg capacity, with300-1500 solid micro-needles per cm² about 150-700 lam tall coated witha drug. The micro-needles penetrate the stratum corneum and remain inthe skin for short duration (e.g., 20 seconds to 15 minutes). In anotherexample, the hollow micro-needle system has up to a 3 mL capacity todeliver liquid formulations using 15-20 microneedles per cm2 beingapproximately 950 μm tall. The micro-needles penetrate the skin to allowthe liquid formulations to flow from the device into the skin. Thehollow micro-needle system may be worn from 1 to 30 minutes depending onthe formulation volume and viscocity.

A device for subcutaneous infusion has been described by Dalton et aland is taught for example in U.S. Pat. No. 7,150,726, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Dalton, multiple needles are incorporated into the device whichdelivers fluid through the needles into a subcutaneous tissue.

A device and a method for intradermal delivery of vaccines and genetherapeutic agents through microcannula have been described by Miksztaet al. and are taught for example in U.S. Pat. No. 7,473,247, thecontents of which are incorporated herein by reference in theirentirety. According to Mitszta, at least one hollow micro-needle isincorporated into the device which delivers the vaccines to thesubject's skin to a depth of between 0.025 mm and 2 mm.

A method of delivering insulin has been described by Pettis et al and istaught for example in U.S. Pat. No. 7,722,595, the contents of which areincorporated herein by reference in their entirety. According to Pettis,two needles are incorporated into a device wherein both needles insertessentially simultaneously into the skin with the first at a depth ofless than 2.5 mm to deliver insulin to intradermal compartment and thesecond at a depth of greater than 2.5 mm and less than 5.0 mm to deliverinsulin to subcutaneous compartment.

Cutaneous injection delivery under suction has been described byKochamba et al. and is taught for example in U.S. Pat. No. 6,896,666,the contents of which are incorporated herein by reference in theirentirety. According to Kochamba, multiple needles in relative adjacencywith each other are incorporated into a device which injects a fluidbelow the cutaneous layer.

A device for withdrawing or delivering a substance through the skin hasbeen described by Down et al and is taught for example in U.S. Pat. No.6,607,513, the contents of which are incorporated herein by reference intheir entirety. According to Down, multiple skin penetrating memberswhich are incorporated into the device have lengths of about 100 micronsto about 2000 microns and are about 30 to 50 gauge.

A device for delivering a substance to the skin has been described byPalmer et al and is taught for example in U.S. Pat. No. 6,537,242, thecontents of which are incorporated herein by reference in theirentirety. According to Palmer, an array of micro-needles is incorporatedinto the device which uses a stretching assembly to enhance the contactof the needles with the skin and provides a more uniform delivery of thesubstance.

A perfusion device for localized drug delivery has been described byZamoyski and is taught for example in U.S. Pat. No. 6,468,247, thecontents of which are incorporated herein by reference in theirentirety. According to Zamoyski, multiple hypodermic needles areincorporated into the device which injects the contents of thehypodermics into a tissue as said hypodermics are being retracted.

A method for enhanced transport of drugs and biological molecules acrosstissue by improving the interaction between micro-needles and human skinhas been described by Prausnitz et al. and is taught for example in U.S.Pat. No. 6,743,211, the contents of which are incorporated herein byreference in their entirety. According to Prausnitz, multiplemicro-needles are incorporated into a device which is able to present amore rigid and less deformable surface to which the micro-needles areapplied.

A device for intraorgan administration of medicinal agents has beendescribed by Ting et al and is taught for example in U.S. Pat. No.6,077,251, the contents of which are incorporated herein by reference intheir entirety. According to Ting, multiple needles having side openingsfor enhanced administration are incorporated into a device which byextending and retracting said needles from and into the needle chamberforces a medicinal agent from a reservoir into said needles and injectssaid medicinal agent into a target organ.

A multiple needle holder and a subcutaneous multiple channel infusionport has been described by Brown and is taught for example in U.S. Pat.No. 4,695,273, the contents of which are incorporated herein byreference in their entirety. According to Brown, multiple needles on theneedle holder are inserted through the septum of the infusion port andcommunicate with isolated chambers in said infusion port.

A dual hypodermic syringe has been described by Horn and is taught forexample in U.S. Pat. No. 3,552,394, the contents of which areincorporated herein by reference in their entirety. According to Horn,two needles incorporated into the device are spaced apart less than 68mm and may be of different styles and lengths, thus enabling injectionsto be made to different depths.

A syringe with multiple needles and multiple fluid compartments has beendescribed by Hershberg and is taught for example in U.S. Pat. No.3,572,336, the contents of which are incorporated herein by reference intheir entirety. According to Hershberg, multiple needles areincorporated into the syringe which has multiple fluid compartments andis capable of simultaneously administering incompatible drugs which arenot able to be mixed for one injection.

A surgical instrument for intradermal injection of fluids has beendescribed by Eliscu et al. and is taught for example in U.S. Pat. No.2,588,623, the contents of which are incorporated herein by reference intheir entirety. According to Eliscu, multiple needles are incorporatedinto the instrument which injects fluids intradermally with a widerdisperse.

An apparatus for simultaneous delivery of a substance to multiple breastmilk ducts has been described by Hung and is taught for example in EP1818017, the contents of which are incorporated herein by reference intheir entirety. According to Hung, multiple lumens are incorporated intothe device which inserts though the orifices of the ductal networks anddelivers a fluid to the ductal networks.

A catheter for introduction of medications to the tissue of a heart orother organs has been described by Tkebuchava and is taught for examplein WO2006138109, the contents of which are incorporated herein byreference in their entirety. According to Tkebuchava, two curved needlesare incorporated which enter the organ wall in a flattened trajectory.

Devices for delivering medical agents have been described by Mckay etal. and are taught for example in WO2006118804, the content of which areincorporated herein by reference in their entirety. According to Mckay,multiple needles with multiple orifices on each needle are incorporatedinto the devices to facilitate regional delivery to a tissue, such asthe interior disc space of a spinal disc.

A method for directly delivering an immunomodulatory substance into anintradermal space within a mammalian skin has been described by Pettisand is taught for example in WO2004020014, the contents of which areincorporated herein by reference in their entirety. According to Pettis,multiple needles are incorporated into a device which delivers thesubstance through the needles to a depth between 0.3 mm and 2 mm.

Methods and devices for administration of substances into at least twocompartments in skin for systemic absorption and improvedpharmacokinetics have been described by Pettis et al. and are taught forexample in WO2003094995, the contents of which are incorporated hereinby reference in their entirety. According to Pettis, multiple needleshaving lengths between about 300 μm and about 5 mm are incorporated intoa device which delivers to intradermal and subcutaneous tissuecompartments simultaneously.

A drug delivery device with needles and a roller has been described byZimmerman et al. and is taught for example in WO2012006259, the contentsof which are incorporated herein by reference in their entirety.According to Zimmerman, multiple hollow needles positioned in a rollerare incorporated into the device which delivers the content in areservoir through the needles as the roller rotates.

Methods and Devices Utilizing Catheters and/or Lumens

Methods and devices using catheters and lumens may be employed toadminister the modified nucleic acids, enhanced modified RNA orribonucleic acids of the present invention on a single, multi- or splitdosing schedule. Such methods and devices are described below.

A catheter-based delivery of skeletal myoblasts to the myocardium ofdamaged hearts has been described by Jacoby et al and is taught forexample in US Patent Publication 20060263338, the contents of which areincorporated herein by reference in their entirety. According to Jacoby,multiple needles are incorporated into the device at least part of whichis inserted into a blood vessel and delivers the cell compositionthrough the needles into the localized region of the subject's heart.

An apparatus for treating asthma using neurotoxin has been described byDeem et al and is taught for example in US Patent Publication20060225742, the contents of which are incorporated herein by referencein their entirety. According to Deem, multiple needles are incorporatedinto the device which delivers neurotoxin through the needles into thebronchial tissue.

A method for administering multiple-component therapies has beendescribed by Nayak and is taught for example in U.S. Pat. No. 7,699,803,the contents of which are incorporated herein by reference in theirentirety. According to Nayak, multiple injection cannulas may beincorporated into a device wherein depth slots may be included forcontrolling the depth at which the therapeutic substance is deliveredwithin the tissue.

A surgical device for ablating a channel and delivering at least onetherapeutic agent into a desired region of the tissue has been describedby McIntyre et al and is taught for example in U.S. Pat. No. 8,012,096,the contents of which are incorporated herein by reference in theirentirety. According to McIntyre, multiple needles are incorporated intothe device which dispenses a therapeutic agent into a region of tissuesurrounding the channel and is particularly well suited fortransmyocardial revascularization operations.

Methods of treating functional disorders of the bladder in mammalianfemales have been described by Versi et al and are taught for example inU.S. Pat. No. 8,029,496, the contents of which are incorporated hereinby reference in their entirety. According to Versi, an array ofmicro-needles is incorporated into a device which delivers a therapeuticagent through the needles directly into the trigone of the bladder.

A device and a method for delivering fluid into a flexible biologicalbarrier have been described by Yeshurun et al. and are taught forexample in U.S. Pat. No. 7,998,119 (device) and U.S. Pat. No. 8,007,466(method), the contents of which are incorporated herein by reference intheir entirety. According to Yeshurun, the micro-needles on the devicepenetrate and extend into the flexible biological barrier and fluid isinjected through the bore of the hollow micro-needles.

A method for epicardially injecting a substance into an area of tissueof a heart having an epicardial surface and disposed within a torso hasbeen described by Bonner et al and is taught for example in U.S. Pat.No. 7,628,780, the contents of which are incorporated herein byreference in their entirety. According to Bonner, the devices haveelongate shafts and distal injection heads for driving needles intotissue and injecting medical agents into the tissue through the needles.

A device for sealing a puncture has been described by Nielsen et al andis taught for example in U.S. Pat. No. 7,972,358, the contents of whichare incorporated herein by reference in their entirety. According toNielsen, multiple needles are incorporated into the device whichdelivers a closure agent into the tissue surrounding the puncture tract.

A method for myogenesis and angiogenesis has been described by Chiu etal. and is taught for example in U.S. Pat. No. 6,551,338, the contentsof which are incorporated herein by reference in their entirety.According to Chiu, 5 to 15 needles having a maximum diameter of at least1.25 mm and a length effective to provide a puncture depth of 6 to 20 mmare incorporated into a device which inserts into proximity with amyocardium and supplies an exogeneous angiogenic or myogenic factor tosaid myocardium through the conduits which are in at least some of saidneedles.

A method for the treatment of prostate tissue has been described byBolmsj et al. and is taught for example in U.S. Pat. No. 6,524,270, thecontents of which are incorporated herein by reference in theirentirety. According to Bolmsj, a device comprising a catheter which isinserted through the urethra has at least one hollow tip extendible intothe surrounding prostate tissue. An astringent and analgesic medicine isadministered through said tip into said prostate tissue.

A method for infusing fluids to an intraosseous site has been describedby Findlay et al. and is taught for example in U.S. Pat. No. 6,761,726,the contents of which are incorporated herein by reference in theirentirety. According to Findlay, multiple needles are incorporated into adevice which is capable of penetrating a hard shell of material coveredby a layer of soft material and delivers a fluid at a predetermineddistance below said hard shell of material.

A device for injecting medications into a vessel wall has been describedby Vigil et al. and is taught for example in U.S. Pat. No. 5,713,863,the contents of which are incorporated herein by reference in theirentirety. According to Vigil, multiple injectors are mounted on each ofthe flexible tubes in the device which introduces a medication fluidthrough a multi-lumen catheter, into said flexible tubes and out of saidinjectors for infusion into the vessel wall.

A catheter for delivering therapeutic and/or diagnostic agents to thetissue surrounding a bodily passageway has been described by Faxon etal. and is taught for example in U.S. Pat. No. 5,464,395, the contentsof which are incorporated herein by reference in their entirety.According to Faxon, at least one needle cannula is incorporated into thecatheter which delivers the desired agents to the tissue through saidneedles which project outboard of the catheter.

Balloon catheters for delivering therapeutic agents have been describedby Orr and are taught for example in WO2010024871, the contents of whichare incorporated herein by reference in their entirety. According toOrr, multiple needles are incorporated into the devices which deliverthe therapeutic agents to different depths within the tissue.

Methods and Devices Utilizing Electrical Current

Methods and devices utilizing electric current may be employed todeliver the modified nucleic acids, enhanced modified RNA or ribonucleicacids of the present invention according to the single, multi- or splitdosing regimens taught herein. Such methods and devices are describedbelow.

An electro collagen induction therapy device has been described byMarquez and is taught for example in US Patent Publication 20090137945,the contents of which are incorporated herein by reference in theirentirety. According to Marquez, multiple needles are incorporated intothe device which repeatedly pierce the skin and draw in the skin aportion of the substance which is applied to the skin first.

An electrokinetic system has been described by Etheredge et al. and istaught for example in US Patent Publication 20070185432, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Etheredge, micro-needles are incorporated into a device which drivesby an electrical current the medication through the needles into thetargeted treatment site.

An iontophoresis device has been described by Matsumura et al. and istaught for example in U.S. Pat. No. 7,437,189, the contents of which areincorporated herein by reference in their entirety. According toMatsumura, multiple needles are incorporated into the device which iscapable of delivering ionizable drug into a living body at higher speedor with higher efficiency.

Intradermal delivery of biologically active agents by needle-freeinjection and electroporation has been described by Hoffmann et al andis taught for example in U.S. Pat. No. 7,171,264, the contents of whichare incorporated herein by reference in their entirety. According toHoffmann, one or more needle-free injectors are incorporated into anelectroporation device and the combination of needle-free injection andelectroporation is sufficient to introduce the agent into cells in skin,muscle or mucosa.

A method for electropermeabilization-mediated intracellular delivery hasbeen described by Lundkvist et al. and is taught for example in U.S.Pat. No. 6,625,486, the contents of which are incorporated herein byreference in their entirety. According to Lundkvist, a pair of needleelectrodes is incorporated into a catheter. Said catheter is positionedinto a body lumen followed by extending said needle electrodes topenetrate into the tissue surrounding said lumen. Then the deviceintroduces an agent through at least one of said needle electrodes andapplies electric field by said pair of needle electrodes to allow saidagent pass through the cell membranes into the cells at the treatmentsite.

A delivery system for transdermal immunization has been described byLevin et al. and is taught for example in WO2006003659, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Levin, multiple electrodes are incorporated into the device whichapplies electrical energy between the electrodes to generate microchannels in the skin to facilitate transdermal delivery.

A method for delivering RF energy into skin has been described bySchomacker and is taught for example in WO2011163264, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Schomacker, multiple needles are incorporated into a device whichapplies vacuum to draw skin into contact with a plate so that needlesinsert into skin through the holes on the plate and deliver RF energy.

DEFINITIONS

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl.

About: As used herein, the term “about” means +/−10% of the recitedvalue.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Auxotrophic: As used herein, the term “auxotrophic” refers to mRNA thatcomprises at least one feature that triggers or induces the degradationor inactivation of the mRNA such that the protein expression issubstantially prevented or reduced in a selected tissue or organ.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety which is capable of or maintains at leasttwo functions. The functions may effect the same outcome or a differentoutcome. The structure that produces the function may be the same ordifferent. For example, bifunctional modified RNAs of the presentinvention may encode a cytotoxic peptide (a first function) while thosenucleosides which comprise the encoding RNA are, in and of themselves,cytotoxic (second function). In this example, delivery of thebifunctional modified RNA to a cancer cell would produce not only apeptide or protein molecule which may ameliorate or treat the cancer butwould also deliver a cytotoxic payload of nucleosides to the cell shoulddegradation, instead of translation of the modified RNA, occur.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological affect on that organism,is considered to be biologically active. In particular embodiments, anucleic acid molecule of the present invention may be consideredbiologically active if even a portion of the nucleic acid molecule isbiologically active or mimics an activity considered biologicallyrelevant.

Chemical terms: The following provides the definition of variouschemical terms from “acyl” to “thiol.”

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,propionyl, butanoyl and the like. Exemplary unsubstituted acyl groupsinclude from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In someembodiments, the alkyl group is further substituted with 1, 2, 3, or 4substituents as described herein.

The term “acylamino,” as used herein, represents an acyl group, asdefined herein, attached to the parent molecular group though an aminogroup, as defined herein (i.e., —N(R^(N1))—C(O)—R, where R is H or anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group and R^(N1) isas defined herein). Exemplary unsubstituted acylamino groups includefrom 1 to 41 carbons (e.g., from 1 to 7, from 1 to 13, from 1 to 21,from 2 to 7, from 2 to 13, from 2 to 21, or from 2 to 41 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “acyloxy,” as used herein, represents an acyl group, as definedherein, attached to the parent molecular group though an oxygen atom(i.e., —O—C(O)—R, where R is H or an optionally substituted C₁₋₆, C₁₋₁₀,or C₁₋₂₀ alkyl group). Exemplary unsubstituted acyloxy groups includefrom 1 to 21 carbons (e.g., from 1 to 7 or from 1 to 11 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “alkaryl,” as used herein, represents an aryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkaryl groups arefrom 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, suchas C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀ alk-C₆₋₁₀ aryl).In some embodiments, the alkylene and the aryl each can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein forthe respective groups. Other groups preceded by the prefix “alk-” aredefined in the same manner, where “alk” refers to a C₁₋₆ alkylene,unless otherwise noted, and the attached chemical structure is asdefined herein.

The term “alkcycloalkyl” represents a cycloalkyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein (e.g., an alkylene group of from 1 to 4, from 1to 6, from 1 to 10, or form 1 to 20 carbons). In some embodiments, thealkylene and the cycloalkyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, andthe like. Alkenyls include both cis and trans isomers. Alkenyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from amino, aryl, cycloalkyl, orheterocyclyl (e.g., heteroaryl), as defined herein, or any of theexemplary alkyl substituent groups described herein.

The term “alkenyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkenyl group (e.g., C₂₋₆ or C₂₋₁₀ alkenyl), unlessotherwise specified. Exemplary alkenyloxy groups include ethenyloxy,propenyloxy, and the like. In some embodiments, the alkenyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “alkheteroaryl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheteroaryl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂heteroaryl, or C₁₋₂₀ alk-C₁₋₁₂ heteroaryl). In some embodiments, thealkylene and the heteroaryl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.Alkheteroaryl groups are a subset of alkheterocyclyl groups.

The term “alkheterocyclyl” represents a heterocyclyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheterocyclyl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heterocyclyl, C₁₋₁₀ alk-C₁₋₁₂heterocyclyl, or C₁₋₂₀ alk-C₁₋₁₂ heterocyclyl). In some embodiments, thealkylene and the heterocyclyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkoxy” represents a chemical substituent of formula —OR,where R is a C₁₋₂₀ alkyl group (e.g., C₁₋₆ or C₁₋₁₀ alkyl), unlessotherwise specified. Exemplary alkoxy groups include methoxy, ethoxy,propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. Insome embodiments, the alkyl group can be further substituted with 1, 2,3, or 4 substituent groups as defined herein (e.g., hydroxy or alkoxy).

The term “alkoxyalkoxy” represents an alkoxy group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkoxy groupsinclude between 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20carbons, such as C₁₋₆ alkoxy-C₁₋₆ alkoxy, C₁₋₁₀ alkoxy-C₁₋₁₀ alkoxy, orC₁₋₂₀ alkoxy-C₁₋₂₀ alkoxy). In some embodiments, the each alkoxy groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkoxyalkyl” represents an alkyl group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups includebetween 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20 carbons,such as C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₁₀ alkoxy-C₁₋₁₀ alkyl, or C₁₋₂₀alkoxy-C₁₋₂₀ alkyl). In some embodiments, the alkyl and the alkoxy eachcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective group.

The term “alkoxycarbonyl,” as used herein, represents an alkoxy, asdefined herein, attached to the parent molecular group through acarbonyl atom (e.g., —C(O)—OR, where R is H or an optionally substitutedC₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplary unsubstitutedalkoxycarbonyl include from 1 to 21 carbons (e.g., from 1 to 11 or from1 to 7 carbons). In some embodiments, the alkoxy group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The term “alkoxycarbonylalkoxy,” as used herein, represents an alkoxygroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., —O-alkyl-C(O)—OR, where R is anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplaryunsubstituted alkoxycarbonylalkoxy include from 3 to 41 carbons (e.g.,from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ alkoxy, C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ alkoxy, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkoxy). Insome embodiments, each alkoxy group is further independently substitutedwith 1, 2, 3, or 4 substituents, as described herein (e.g., a hydroxygroup).

The term “alkoxycarbonylalkyl,” as used herein, represents an alkylgroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., -alkyl-C(O)—OR, where R is an optionallysubstituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group). Exemplary unsubstitutedalkoxycarbonylalkyl include from 3 to 41 carbons (e.g., from 3 to 10,from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31 carbons, suchas C₁₋₆ alkoxycarbonyl-C₁₋₆ alkyl, C₁₋₁₀ alkoxycarbonyl-C₁₋₁₀ alkyl, orC₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkyl). In some embodiments, each alkyl andalkoxy group is further independently substituted with 1, 2, 3, or 4substituents as described herein (e.g., a hydroxy group).

The term “alkyl,” as used herein, is inclusive of both straight chainand branched chain saturated groups from 1 to 20 carbons (e.g., from 1to 10 or from 1 to 6), unless otherwise specified. Alkyl groups areexemplified by methyl, ethyl, n- and isopropyl, n-, sec-, iso- andtert-butyl, neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as definedherein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino(i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8)hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(1′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein. For example, the alkylene group of aC₁-alkaryl can be further substituted with an oxo group to afford therespective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term“C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylenegroups having between x and y carbons. Exemplary values for x are 1, 2,3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, orC₂₋₂₀ alkylene). In some embodiments, the alkylene can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein foran alkyl group.

The term “alkylsulfinyl,” as used herein, represents an alkyl groupattached to the parent molecular group through an —S(O)— group.Exemplary unsubstituted alkylsulfinyl groups are from 1 to 6, from 1 to10, or from 1 to 20 carbons. In some embodiments, the alkyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein.

The term “alkylsulfinylalkyl,” as used herein, represents an alkylgroup, as defined herein, substituted by an alkylsulfinyl group.Exemplary unsubstituted alkylsulfinylalkyl groups are from 2 to 12, from2 to 20, or from 2 to 40 carbons. In some embodiments, each alkyl groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

The term “alkynyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkynyl group (e.g., C₂₋₆ or C₂₋₁₀ alkynyl), unlessotherwise specified. Exemplary alkynyloxy groups include ethynyloxy,propynyloxy, and the like. In some embodiments, the alkynyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “amidine,” as used herein, represents a —C(═NH)NH₂ group.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl, sulfoalkyl,heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g.,alkheteroaryl), wherein each of these recited R^(N1) groups can beoptionally substituted, as defined herein for each group; or two R^(N1)combine to form a heterocyclyl or an N-protecting group, and whereineach R^(N2) is, independently, H, alkyl, or aryl. The amino groups ofthe invention can be an unsubstituted amino (i.e., —NH₂) or asubstituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, aminois —NH₂ or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl,sulfoalkyl, or aryl, and each R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). In some embodiments, the amino acid isattached to the parent molecular group by a carbonyl group, where theside chain or amino group is attached to the carbonyl group. Exemplaryside chains include an optionally substituted alkyl, aryl, heterocyclyl,alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.Exemplary amino acids include alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline,ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine,taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groupsmay be optionally substituted with one, two, three, or, in the case ofamino acid groups of two carbons or more, four substituentsindependently selected from the group consisting of: (1) C₁₋₆ alkoxy;(2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy;(9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(H′)R^(G′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆-10 aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(F)C(O)OR^(K′), wherein R^(J′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aminoalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aminoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, indenyl, and the like, and may be optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from the groupconsisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy;(14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy);(17) —(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, andR^(A′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b)C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integer fromzero to four and where R^(D′) is selected from the group consisting of(a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁-6 alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The term “arylalkoxy,” as used herein, represents an alkaryl group, asdefined herein, attached to the parent molecular group through an oxygenatom. Exemplary unsubstituted alkoxyalkyl groups include from 7 to 30carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₆₋₁₀aryl-C₁₋₆ alkoxy, C₆₋₁₀ aryl-C₁₋₁₀ alkoxy, or C₆₋₁₀ aryl-C₁₋₂₀ alkoxy).In some embodiments, the arylalkoxy group can be substituted with 1, 2,3, or 4 substituents as defined herein

The term “aryloxy” represents a chemical substituent of formula —OR′,where R′ is an aryl group of 6 to 18 carbons, unless otherwisespecified. In some embodiments, the aryl group can be substituted with1, 2, 3, or 4 substituents as defined herein.

The term “aryloyl,” as used herein, represents an aryl group, as definedherein, that is attached to the parent molecular group through acarbonyl group. Exemplary unsubstituted aryloyl groups are of 7 to 11carbons. In some embodiments, the aryl group can be substituted with 1,2, 3, or 4 substituents as defined herein.

The term “azido” represents an —N₃ group, which can also be representedas —N═N═N.

The term “bicyclic,” as used herein, refer to a structure having tworings, which may be aromatic or non-aromatic. Bicyclic structuresinclude spirocyclyl groups, as defined herein, and two rings that shareone or more bridges, where such bridges can include one atom or a chainincluding two, three, or more atoms. Exemplary bicyclic groups include abicyclic carbocyclyl group, where the first and second rings arecarbocyclyl groups, as defined herein; a bicyclic aryl groups, where thefirst and second rings are aryl groups, as defined herein; bicyclicheterocyclyl groups, where the first ring is a heterocyclyl group andthe second ring is a carbocyclyl (e.g., aryl) or heterocyclyl (e.g.,heteroaryl) group; and bicyclic heteroaryl groups, where the first ringis a heteroaryl group and the second ring is a carbocyclyl (e.g., aryl)or heterocyclyl (e.g., heteroaryl) group. In some embodiments, thebicyclic group can be substituted with 1, 2, 3, or 4 substituents asdefined herein for cycloalkyl, heterocyclyl, and aryl groups.

The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to anoptionally substituted C₃₋₁₂ monocyclic, bicyclic, or tricyclicstructure in which the rings, which may be aromatic or non-aromatic, areformed by carbon atoms. Carbocyclic structures include cycloalkyl,cycloalkenyl, and aryl groups.

The term “carbamoyl,” as used herein, represents —C(O)—N(R^(N1))₂, wherethe meaning of each R^(N1) is found in the definition of “amino”provided herein.

The term “carbamoylalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carbamoyl group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “carbamyl,” as used herein, refers to a carbamate group havingthe structure —NR^(N1)C(═O)OR or —OC(═O)N(R^(N1))₂, where the meaning ofeach R^(N1) is found in the definition of “amino” provided herein, and Ris alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl (e.g.,heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as definedherein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxyaldehyde” represents an acyl group having the structure—CHO.

The term “carboxy,” as used herein, means —CO₂H.

The term “carboxyalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkoxy group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein for the alkyl group.

The term “carboxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkoxy” represents a chemical substituent of formula —OR,where R is a C₃₋₈ cycloalkyl group, as defined herein, unless otherwisespecified. The cycloalkyl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein. Exemplary unsubstitutedcycloalkoxy groups are from 3 to 8 carbons. In some embodiment, thecycloalkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl,and the like. When the cycloalkyl group includes one carbon-carbondouble bond, the cycloalkyl group can be referred to as a “cycloalkenyl”group. Exemplary cycloalkenyl groups include cyclopentenyl,cyclohexenyl, and the like. The cycloalkyl groups of this invention canbe optionally substituted with: (1) C₁₋₇ acyl (e.g., carboxyaldehyde);(2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl,(carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g., perfluoroalkyl),hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl);(3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such as perfluoroalkoxy); (4) C₁₋₆alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8)azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo;(12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g.,C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q is an integer fromzero to four, and R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl;(18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to fourand where R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d)C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integerfrom zero to four and where R^(D′) is selected from the group consistingof (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁-12 heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “effective amount” of an agent, as used herein, is that amountsufficient to effect beneficial or desired results, for example,clinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that treats cancer, an effective amount of anagent is, for example, an amount sufficient to achieve treatment, asdefined herein, of cancer, as compared to the response obtained withoutadministration of the agent.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “haloalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkoxy may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkoxy groupsinclude perfluoroalkoxys (e.g., —OCF₃), —OCHF₂, —OCH₂F, —OCCl₃,—OCH₂CH₂Br, —OCH₂CH(CH₂CH₂Br)CH₃, and —OCHICH₃. In some embodiments, thehaloalkoxy group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “haloalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkyl may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkyl groupsinclude perfluoroalkyls (e.g., —CF₃), —CHF₂, —CH₂F, —CCl₃, —CH₂CH₂Br,—CH₂CH(CH₂CH₂Br)CH₃, and —CHICH₃. In some embodiments, the haloalkylgroup can be further substituted with 1, 2, 3, or 4 substituent groupsas described herein for alkyl groups.

The term “heteroalkylene,” as used herein, refers to an alkylene group,as defined herein, in which one or two of the constituent carbon atomshave each been replaced by nitrogen, oxygen, or sulfur. In someembodiments, the heteroalkylene group can be further substituted with 1,2, 3, or 4 substituent groups as described herein for alkylene groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like. Examples of fused heterocyclyls includetropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics includepyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl,morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where

E′ is selected from the group consisting of —N— and —CH—; F′ is selectedfrom the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—, —CH═N—,—CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—, —O—, and—S—; and G′ is selected from the group consisting of —CH— and —N—. Anyof the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇ acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and R^(P) is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “(heterocyclyl)imino,” as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an imino group. In some embodiments, the heterocyclylgroup can be substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “(heterocyclyl)oxy,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group throughan oxygen atom. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “(heterocyclyl)oyl,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group througha carbonyl group. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxy,” as used herein, represents an —OH group.

The term “hydroxyalkenyl,” as used herein, represents an alkenyl group,as defined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by dihydroxypropenyl,hydroxyisopentenyl, and the like.

The term “hydroxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by hydroxymethyl,dihydroxypropyl, and the like.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups, such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “nitro,” as used herein, represents an —NO₂ group.

The term “oxo” as used herein, represents ═O.

The term “perfluoroalkyl,” as used herein, represents an alkyl group, asdefined herein, where each hydrogen radical bound to the alkyl group hasbeen replaced by a fluoride radical. Perfluoroalkyl groups areexemplified by trifluoromethyl, pentafluoroethyl, and the like.

The term “perfluoroalkoxy,” as used herein, represents an alkoxy group,as defined herein, where each hydrogen radical bound to the alkoxy grouphas been replaced by a fluoride radical. Perfluoroalkoxy groups areexemplified by trifluoromethoxy, pentafluoroethoxy, and the like.

The term “spirocyclyl,” as used herein, represents a C₂₋₇ alkylenediradical, both ends of which are bonded to the same carbon atom of theparent group to form a spirocyclic group, and also a C₁₋₆ heteroalkylenediradical, both ends of which are bonded to the same atom. Theheteroalkylene radical forming the spirocyclyl group can containing one,two, three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. In some embodiments, thespirocyclyl group includes one to seven carbons, excluding the carbonatom to which the diradical is attached. The spirocyclyl groups of theinvention may be optionally substituted with 1, 2, 3, or 4 substituentsprovided herein as optional substituents for cycloalkyl and/orheterocyclyl groups.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

The term “sulfoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a sulfo group of —SO₃H. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thioalkaryl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkaryl group. In someembodiments, the alkaryl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein.

The term “thioalkheterocyclyl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkheterocyclyl group. In someembodiments, the alkheterocyclyl group can be further substituted with1, 2, 3, or 4 substituent groups as described herein.

The term “thioalkoxy,” as used herein, represents a chemical substituentof formula —SR, where R is an alkyl group, as defined herein. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “thiol” represents an —SH group.

Compound: As used herein, the term “compound,” as used herein, is meantto include all stereoisomers, geometric isomers, tautomers, and isotopesof the structures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond together with the concomitant migration of aproton. Tautomeric forms include prototropic tautomers which areisomeric protonation states having the same empirical formula and totalcharge. Example prototropic tautomers include ketone-enol pairs,amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs,enamine-imine pairs, and annular forms where a proton can occupy two ormore positions of a heterocyclic system, for example, 1H- and3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium orsterically locked into one form by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an oligonucleotide orpolypeptide or may apply to a portion, region or feature thereof.Conservation of sequence may apply to the entire length of anoligonucleotide or polypeptide or may apply to a portion, region orfeature thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of a modifiednucleic acid to targeted cells.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Device: As used herein, the term “device” means a piece of equipmentdesigned to serve a special purpose. The device may comprise manyfeatures such as, but not limited to, components, electrical (e.g.,wiring and circuits), storage modules and analysis modules.

Disease: As used herein, the term “disease” refers to an abnormalcondition affecting the body of an organism often showing specificbodily symptoms.

Disorder: As used herein, the term “disorder,” refers to a disruption ofor an interference with normal functions or established systems of thebody.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Encoded protein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence which encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Exosome: As used herein, “exosome” is a vesicle secreted by mammaliancells or a complex involved in RNA degradation.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least amodified nucleic acid and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Heterologous: As used herein, the term “heterologous” in reference to anuntranslated region such as a 5′UTR or 3′UTR means a region of nucleicacid, particularly untranslated nucleic acid which is not naturallyfound with the coding region encoded on the same or instantpolynucleotide, primary construct or mmRNA. Homologous UTRs for examplewould represent those UTRs which are naturally found associated with thecoding region of the mRNA, such as the wild type UTR.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% similar. The term “homologous” necessarily refers to acomparison between at least two sequences (polynucleotide or polypeptidesequences).

In accordance with the invention, two polynucleotide sequences areconsidered to be homologous if the polypeptides they encode are at leastabout 50% identical, at least about 60% identical, at least about 70%identical, at least about 80% identical, or at least about 90% identicalfor at least one stretch of at least about 20 amino acids.

In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the invention, two protein sequences are considered tobe homologous if the proteins are at least about 50% identical, at leastabout 60% identical, at least about 70% identical, at least about 80%identical, or at least about 90% identical for at least one stretch ofat least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between oligonucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Atschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof. Methods forisolating compounds and their salts are routine in the art.

Linker: As used herein, a linker refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker may be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form modified mRNA multimers (e.g.,through linkage of two or more modified nucleic acids) or modified mRNAconjugates, as well as to administer a payload, as described herein.Examples of chemical groups that can be incorporated into the linkerinclude, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino,ether, thioether, ester, alkylene, heteroalkylene, aryl, orheterocyclyl, each of which can be optionally substituted, as describedherein. Examples of linkers include, but are not limited to, unsaturatedalkanes, polyethylene glycols (e.g., ethylene or propylene glycolmonomeric units, e.g., diethylene glycol, dipropylene glycol,triethylene glycol, tripropylene glycol, tetraethylene glycol, ortetraethylene glycol), and dextran polymers, Other examples include, butare not limited to, cleavable moieties within the linker, such as, forexample, a disulfide bond (—S—S—) or an azo bond (—N═N—), which can becleaved using a reducing agent or photolysis. Non-limiting examples of aselectively cleavable bond include an amido bond can be cleaved forexample by the use of tris(2-carboxyethyl)phosphine (TCEP), or otherreducing agents, and/or photolysis, as well as an ester bond can becleaved for example by acidic or basic hydrolysis.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the invention. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present invention are modified bythe introduction of non-natural nucleosides and/or nucleotides.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g. alkyl) per se is optional. Peptide: Asused herein, “peptide” is less than or equal to 50 amino acids long,e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Pharmaceutical composition: The phrase “pharmaceutical composition”refers to a composition that alters the etiology of a disease, disorderand/or condition.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977),each of which is incorporated herein by reference in its entirety.

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestested in ssomeway and which release or are converted into the active drug moiety priorto, upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Pseudouridine: As used herein, pseudouridine refers to the C-glycosideisomer of the nucleoside uridine. A “pseudouridine analog” is anymodification, variant, isoform or derivative of pseudouridine. Forexample, pseudouridine analogs include but are not limited to1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine,1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine,1-methyl-pseudouridine (m¹ψ), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine,1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ), and2′-O-methyl-pseudouridine (ψm).

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Reducing the effect: As used herein, the phrase “reducing the effect”when referring to symptoms, means reducing, eliminating or alleviatingthe symptom in the subject. It does not necessarily mean that thesymptom will, in fact, be completely eliminated, reduced or alleviated.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Seed: As used herein with respect to micro RNA (miRNA), a miRNA “seed”is a sequence with nucleotide identity at positions 2-8 of the maturemiRNA. In one embodiment, a miRNA seed comprises positions 2-7 of themature miRNA.

Side effect: As used herein, the phrase “side effect” refers to asecondary effect of treatment.

Signal Peptide Sequences: As used herein, the phrase “signal peptidesequences” refers to a sequence which can direct the transport orlocalization of a protein.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 15 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Symptom: As used herein, the term “symptom” is a signal of a disease,disorder and/or condition. For example, symptoms may be felt or noticedby the subject who has them but may not be easily accessed by looking ata subject's outward appearance or behaviors. Examples of symptomsinclude, but are not limited to, weakness, aches and pains, fever,fatigue, weight loss, blood clots, increased blood calcium levels, lowwhite blood cell count, short of breath, dizziness, headaches,hyperpigmentation, jaundice, erthema, pruritis, excessive hair growth,change in bowel habits, change in bladder function, long-lasting sores,white patches inside the mouth, white spots on the tongue, unusualbleeding or discharge, thickening or lump on parts of the body,indigestion, trouble swallowing, changes in warts or moles, change innew skin and nagging cough or hoarseness.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Terminal region: As used herein, the term “terminal region” refers to aregion on the 5′ or 3′ end of a region of linked nucleosides encoding apolypeptide of interest or coding region.

Terminally optimized: The term “terminally optimized” when referring tonucleic acids means the terminal regions of the nucleic acid areimproved over the native terminal regions.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to a disease, disorder,and/or condition, to treat, improve symptoms of, diagnose, prevent,and/or delay the onset of the disease, disorder, and/or condition.

Therapeutically effective outcome: As used herein, “therapeuticallyeffective amount” means an amount of an agent to be delivered (e.g.,nucleic acid, drug, therapeutic agent, diagnostic agent, prophylacticagent, etc.) that is sufficient, when administered to a subjectsuffering from or susceptible to a disease, disorder, and/or condition,to treat, improve symptoms of, diagnose, prevent, and/or delay the onsetof the disease, disorder, and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particulardisease, disorder, and/or condition. For example, “treating” cancer mayrefer to inhibiting survival, growth, and/or spread of a tumor.Treatment may be administered to a subject who does not exhibit signs ofa disease, disorder, and/or condition and/or to a subject who exhibitsonly early signs of a disease, disorder, and/or condition for thepurpose of decreasing the risk of developing pathology associated withthe disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits the inclusion of additional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

EXAMPLES Example 1 Modified mRNA Production

Modified mRNAs according to the invention are made using standardlaboratory methods and materials.

The open reading frame with various upstream or downstream regions(β-globin, tags, etc.) is ordered from DNA2.0 (Menlo Park, Calif.) andtypically contains a multiple cloning site with XbaI recognition. Uponreceipt of the construct, it is reconstituted and transformed intochemically competent E. coli. For the present invention, NEB DH5-alphaCompetent E. coli are used. A typical clone map is shown in FIG. 3.Transformations are performed according to NEB instructions using 100 ngof plasmid. The protocol is as follows:

1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10minutes.

2. Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture.Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

3. Place the mixture on ice for 30 minutes. Do not mix.

4. Heat shock at 42° C. for exactly 30 seconds. Do not mix.

5. Place on ice for 5 minutes. Do not mix.

6. Pipette 950 μl of room temperature SOC into the mixture.

7. Place at 37° C. for 60 minutes. Shake vigorously (250 rpm) or rotate.

8. Warm selection plates to 37° C.

9. Mix the cells thoroughly by flicking the tube and inverting.

10. Spread 50-100 μl of each dilution onto a selection plate andincubate overnight at 37° C. Alternatively, incubate at 30° C. for 24-36hours or 25° C. for 48 hours.

A single colony is then used to inoculate 5 ml of LB growth media usingthe appropriate antibiotic and then allowed to grow (250 RPM, 37° C.)for 5 hours. This is then used to inoculate a 200 ml culture medium andallowed to grow overnight under the same conditions.

To isolate the plasmid (up to 850 μg), a maxi prep is performed usingthe Invitrogen PureLink™ HiPure Maxiprep Kit (Carlsbad, Calif.),following the manufacturer's instructions.

In order to generate cDNA for In Vitro Transcription (IVT), the plasmidis first linearized using a restriction enzyme such as XbaI. A typicalrestriction digest with XbaI will comprise the following: Plasmid 1.0μg; 10× Buffer 1.0 μl; XbaI 1.5 μl; dH₂0

Up to 10 μl; incubated at 37° C. for 1 hr. If performing at lab scale(<5 μg), the reaction is cleaned up using Invitrogen's PureLink™ PCRMicro Kit (Carlsbad, Calif.) per manufacturer's instructions. Largerscale purifications may need to be done with a product that has a largerload capacity such as Invitrogen's standard PureLink PCR Kit (Carlsbad,Calif.). Following the cleanup, the linearized vector is quantifiedusing the NanoDrop and analyzed to confirm linearization using agarosegel electrophoresis.

As a non-limiting example, G-CSF may represent the polypeptide ofinterest. Sequences used in the steps outlined in Examples 1-5 are shownin Table 16. It should be noted that the start codon (ATG) has beenunderlined in each sequence of Table 16.

TABLE 16 G-CSF Sequences SEQ ID NO Description 4255 cDNAsequence:ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGA 4256cDNA having T7 polymerase site, AfeI and Xba restriction site:TAATACGACTCACTATA GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCG GCCGCTCGAGCATGCATCTAGA 4257Optimized sequence; containing T7 polymerasesite, AfeI and Xba restriction site TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCG GCCGCTCGAGCATGCATCTAGA 4258mRNA sequence (transcribed) GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCU GAGUAGGAAG

Example 2 PCR for cDNA Production

PCR procedures for the preparation of cDNA is performed using 2×KAPAHiFi™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix 12.5 μl; Forward Primer (10 uM) 0.75 μl;Reverse Primer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH₂0 dilutedto 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25cycles of 98° C. 20 sec, then 58° C. 15 sec, then 72° C. 45 sec, then72° C. 5 min. then 4° C. to termination.

The reverse primer of the instant invention incorporates a poly-T₁₂₀ fora poly-A₁₂₀ in the mRNA. Other reverse primers with longer or shorterpoly(T) tracts can be used to adjust the length of the poly(A) tail inthe mRNA.

The reaction is cleaned up using Invitrogen's PureLink™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 3 In Vitro Transcription

The in vitro transcription reaction generates mRNA containing modifiednucleotides or modified RNA. The input nucleotide triphosphate (NTP) mixis made in-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

1. Template cDNA 1.0 μg

2. 10× transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM MgCl₂, 50 mMDTT, 10 mM Spermidine) 2.0 μl

3. Custom NTPs (25 mM each) 7.2 μl

4. RNase Inhibitor20 U

5. T7 RNA polymerase 3000 U

6. dH₂0 Up to 20.0 μl. and

7. Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGAclear™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 4 Enzymatic Capping of mRNA

Capping of the mRNA is performed as follows where the mixture includes:IVT RNA 60 μg-180 μg and dH₂0 up to 72 μl. The mixture is incubated at65° C. for 5 minutes to denature RNA, then transfer immediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl2)(10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400 U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂0 (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The mRNA is then purified using Ambion's MEGAclear™ Kit (Austin, Tex.)following the manufacturer's instructions. Following the cleanup, theRNA is quantified using the NanoDrop (ThermoFisher, Waltham, Mass.) andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred. The RNA productmay also be sequenced by running a reverse-transcription-PCR to generatethe cDNA for sequencing.

Example 5 PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl2)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂0 up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGAclear™ kit (up to 500 μg). Poly-A Polymerase is preferablya recombinant enzyme expressed in yeast.

For studies performed and described herein, the poly-A tail is encodedin the IVT template to comprise 160 nucleotides in length. However, itshould be understood that the processivity or integrity of the polyAtailing reaction may not always result in exactly 160 nucleotides. HencepolyA tails of approximately 160 nucleotides, e.g, about 150-165, 155,156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scopeof the invention.

Example 6 Natural 5′ Caps and 5′ Cap Analogues

5′-capping of modified RNA may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3″-O-Me-m7G(5′)ppp(5′)G; G(5′)ppp(5′)A;G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). 5′-capping of modified RNA may be completedpost-transcriptionally using a Vaccinia Virus Capping Enzyme to generatethe “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs, Ipswich,Mass.). Cap 1 structure may be generated using both Vaccinia VirusCapping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs may have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 7 Chemical Cap vs. Enzymatically-Derived Cap Protein ExpressionAssay

Synthetic mRNAs encoding human G-CSF containing the ARCA cap analog orthe Cap1 structure can be transfected into human primary keratinocytesat equal concentrations. 6, 12, 24 and 36 hours post-transfection theamount of G-CSF secreted into the culture medium can be assayed byELISA. Synthetic mRNAs that secrete higher levels of G-CSF into themedium would correspond to a synthetic mRNA with a highertranslationally-competent Cap structure.

Example 8 Chemical Cap vs. Enzymatically-Derived Cap Purity Analysis

Synthetic mRNAs encoding human G-CSF containing the ARCA cap analog orthe Cap1 structure crude synthesis products can be compared for purityusing denaturing Agarose-Urea gel electrophoresis or HPLC analysis.Synthetic mRNAs with a single, consolidated band by electrophoresiscorrespond to the higher purity product compared to a synthetic mRNAwith multiple bands or streaking bands. Synthetic mRNAs with a singleHPLC peak would also correspond to a higher purity product. The cappingreaction with a higher efficiency would provide a more pure mRNApopulation.

Example 9 Chemical Cap Vs. Enzymatically-Derived Cap Cytokine Analysis

Synthetic mRNAs encoding human G-CSF containing the ARCA cap analog orthe Cap1 structure can be transfected into human primary keratinocytesat multiple concentrations. 6, 12, 24 and 36 hours post-transfection theamount of pro-inflammatory cytokines such as TNF-alpha and IFN-betasecreted into the culture medium can be assayed by ELISA. SyntheticmRNAs that secrete higher levels of pro-inflammatory cytokines into themedium would correspond to a synthetic mRNA containing animmune-activating cap structure.

Example 10 Chemical Cap Vs. Enzymatically-Derived Cap Capping ReactionEfficiency

Synthetic mRNAs encoding human G-CSF containing the ARCA cap analog orthe Cap1 structure can be analyzed for capping reaction efficiency byLC-MS after capped mRNA nuclease treatment. Nuclease treatment of cappedmRNAs would yield a mixture of free nucleotides and the capped5′-5-triphosphate cap structure detectable by LC-MS. The amount ofcapped product on the LC-MS spectra can be expressed as a percent oftotal mRNA from the reaction and would correspond to capping reactionefficiency. The Cap structure with a higher capping reaction efficiencywould have a higher amount of capped product by LC-MS.

Example 11 Agarose Gel Electrophoresis of Modified RNA or RT PCRProducts

Individual modRNAs (200-400 ng in a 20 μl volume) or reverse transcribedPCR products (200-400 ng) are loaded into a well on a non-denaturing1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and run for 12-15minutes according to the manufacturer protocol.

Example 12 Nanodrop Modified RNA Quantification and UV Spectral Data

Modified RNAs in TE buffer (1 μl) are used for Nanodrop UV absorbancereadings to quantitate the yield of each modified RNA from an in vitrotranscription reaction.

Example 13 In Vitro Transcription of Modified RNA Containing VaryingPoly-A Tail Lengths

Modified mRNAs were made using standard laboratory methods and materialsfor in vitro transcription with the exception that the nucleotide mixcontains modified nucleotides. Modified mRNAs of the present exampleincluded 5-methycytosine and pseudouridine. The open reading frame (ORF)of the gene of interest is flanked by a 5′ untranslated region (UTR)containing a strong Kozak translational initiation signal and analpha-globin 3′ UTR terminating with an oligo(dT) sequence for templatedaddition of a polyA tail for modified RNAs not incorporating Adenosineanalogs. Adenosine-containing modRNAs are synthesized without an oligo(dT) sequence to allow for post-transcription poly (A) polymerasepoly-(A) tailing. Poly-a tail lengths of 0 nts, 80 nts, 120 nts, 160 ntswere generated for human G-CSF. G-CSF sequences include the cDNAsequence (SEQ ID NO: 4257), the mRNA sequence (SEQ ID NO: 4258) and theprotein sequence (SEQ ID NO: 4259). Detection of G-CSF may be performedby the primer probe sets for cDNA including the forward primer TTG GACCCT CGT ACA GAA GCT AAT ACG (SEQ ID NO: 4260), a reverse primer fortemplate Poly(A) tailing T(₁₂₀)CT TCC TAC TCA GGC TTT ATT CAA AGA CCA(SEQ ID NO: 4261) and a reverse primer for post-transcriptional Poly(A)polymerase tailing CTT CCT ACT CAG GCT TTA TTC AAA GAC CA (SED ID NO:4262). Detection may also be performed by G-CSF modified nucleic acidmolecule reverse-transcriptase polymerase chain reaction (RT-PCR)forward primer TGG CCG GTC CCG CGA CCC AA (SEQ ID NO: 4263) and reverseprimer GCT TCA CGG CTG CGC AAG AT (SEQ ID NO: 4264).

Synthesized reverse primers were designed and ordered from IDT. Thereverse primers incorporate a poly-T40, poly-T80, poly-T120, poly-T160for a poly-A40, poly-A80, poly-A120, and poly-A160 respectively. TheHuman Embryonic Kidney (HEK) 293 were grown in Eagles' Minimal EssentialMedium (EMEM) and 10% Fetal Bovine Serum (FBS) until they reached aconfluence of 80-90%. Approximately 80,000 cells were transfected with100 ng and 500 ng of modified RNA complexed with RNAiMax from Invitrogen(Carlsbad, Calif.) in a 24-well plate. The RNA:RNAiMax complex wasformed by first incubating the RNAiMax with EMEM in a 5× volumetricdilution for 10 minutes at room temperature.

The RNA vial was then mixed with the RNAiMAX vial and incubated for20-30 at room temperature before being added to the cells in a drop-wisefashion. Recombinant Human G-CSF was added at 2 ng/mL to the controlcell culture wells. The concentration of secreted Human G-CSF wasmeasured at 12 hours post-transfection. FIG. 4 shows the histogram forthe Enzyme-linked immunosorbent assay (ELISA) for Human G-CSF fromHEK293 cells transfected with human G-CSF modified RNA that had varyingpoly-A tail lengths: 0 nts, 80 nts, 120 nts, 160 nts. We observedincreased protein expression with the 160 nts poly-A tail.

From the data it can be determined that longer poly-A tails produce moreprotein and that this activity is dose dependent.

Example 14 Expression of Modified Nucleic Acid with microRNA BindingSite

Human embryonic kidney epithelial cells (HEK293A) and primary humanhepatocytes (Hepatocytes) were seeded at a density of 200,000 per wellin 500 ul cell culture medium (InVitro GRO medium from Celsis, Chicago,Ill.). G-CSF mRNA having an alpha-globin 3′UTR (G-CSF alpha) (cDNAsequence is shown in SEQ ID NO: 4265; mRNA sequence is shown in SEQ IDNO: 4266; polyA tail of approximately 160 nucleotides not shown insequence; 5′Cap, Cap1; fully modified with 5-methylcytidine andpseudouridine) G-CSF mRNA having an alpha-globin 3′UTR and a miR-122binding site (G-CSF miR-122) (cDNA sequence is shown in SEQ ID NO: 4267;mRNA sequence is shown in SEQ ID NO: 4268; polyA tail of approximately160 nucleotides not shown in sequence; 5′Cap, Cap1; fully modified with5-methylcytidine and pseudouridine) or G-CSF mRNA having an alpha-globin3′UTR with four miR-122 binding sites with the seed deleted (G-CSF noseed) (cDNA sequence is shown in SEQ ID NO: 4269; mRNA sequence is shownin SEQ ID NO: 4270; polyA tail of approximately 160 nucleotides notshown in sequence; 5′Cap, Cap1; fully modified with 5-methylcytidine andpseudouridine) was tested at a concentration of 250 ng per well in 24well plates. The expression of G-CSF was measured by ELISA and theresults are shown in Table 17

TABLE 17 miR-122 Binding Sites HEK293A Hepatocytes Protein ExpressionProtein Expression (ng/mL) (ng/mL) G-CSF alpha 99.85 8.18 G-CSF miR-12287.67 0 G-CSF no seed 200.2 8.05

Since HEK293 cells do not express miR-122 there was no down-regulationof G-CSF protein from the sequence containing miR-122. Whereas, thehuman hepatocytes express high levels of miR-122 and there was a drasticdown-regulation of G-CSF protein observed when the G-CSF sequencecontained the miR-122 target sequence. Consequently, the mRNA functionedas an auxotrophic mRNA.

Example 15 Directed SAR of Pseudouridine and N1-methyl PseudoUridine

With the recent focus on the pyrimidine nucleoside pseudouridine, aseries of structure-activity studies were designed to investigate mRNAcontaining modifications to pseudouridine or N1-methyl-pseudourdine.

The study was designed to explore the effect of chain length, increasedlipophilicity, presence of ring structures, and alteration ofhydrophobic or hydrophilic interactions when modifications were made atthe N1 position, C6 position, the 2-position, the 4-position and on thephosphate backbone. Stability is also investigated.

To this end, modifications involving alkylation, cycloalkylation,alkyl-cycloalkylation, arylation, alkyl-arylation, alkylation moietieswith amino groups, alkylation moieties with carboxylic acid groups, andalkylation moieties containing amino acid charged moieties areinvestigated. The degree of alkylation is generally C₁-C₆. Examples ofthe chemistry modifications include those listed in Table 18 and 19.

TABLE 18 Pseudouridine and N1-methyl Pseudo Uridine SAR NaturallyChemistry Modification Compound # occuring N1-ModificationsN1-Ethyl-pseudo-UTP 1 N N1-Propyl-pseudo-UTP 2 NN1-iso-propyl-pseudo-UTP 3 N N1-(2,2,2-Trifluoroethyl)-pseudo-UTP 4 NN1-Cyclopropyl-pseudo-UTP 5 N N1-Cyclopropylmethyl-pseudo-UTP 6 NN1-Phenyl-pseudo-UTP 7 N N1-Benzyl-pseudo-UTP 8 NN1-Aminomethyl-pseudo-UTP 9 N P seudo-UTP-N1-2-ethanoic acid 10 NN1-(3-Amino-3-carboxypropyl)pseudo-UTP 11 NN1-Methyl-3-(3-amino-3-carboxypropyl) 12 Y pseudo-UTP C-6 Modifications6-Methyl-pseudo-UTP 13 N 6-Trifluoromethyl-pseudo-UTP 14 N6-Methoxy-pseudo-UTP 15 N 6-Phenyl-pseudo-UTP 16 N 6-Iodo-pseudo-UTP 17N 6-Bromo-pseudo-UTP 18 N 6-Chloro-pseudo-UTP 19 N 6-Fluoro-pseudo-UTP20 N 2- or 4-position Modifications 4-Thio-pseudo-UTP 21 N2-Thio-pseudo-UTP 22 N Phosphate backbone ModificationsAlpha-thio-pseudo-UTP 23 N N1-Me-alpha-thio-pseudo-UTP 24 N

TABLE 19 Pseudouridine and N1-methyl Pseudo Uridine SAR NaturallyChemistry Modification Compound # occuring N1-Methyl-pseudo-UTP  1 YN1-Butyl-pseudo-UTP  2 N N1-tert-Butyl-pseudo-UTP  3 NN1-Pentyl-pseudo-UTP  4 N N1-Hexyl-pseudo-UTP  5 NN1-Trifluoromethyl-pseudo-UTP  6 Y N1-Cyclobutyl-pseudo-UTP  7 NN1-Cyclopentyl-pseudo-UTP  8 N N1-Cyclohexyl-pseudo-UTP  9 NN1-Cycloheptyl-pseudo-UTP 10 N N1-Cyclooctyl-pseudo-UTP 11 NN1-Cyclobutylmethyl-pseudo-UTP 12 N N1-Cyclopentylmethyl-pseudo-UTP 13 NN1-Cyclohexylmethyl-pseudo-UTP 14 N N1-Cycloheptylmethyl-pseudo-UTP 15 NN1-Cyclooctylmethyl-pseudo-UTP 16 N N1-p-tolyl-pseudo-UTP 17 NN1-(2,4,6-Trimethyl-phenyl)pseudo-UTP 18 NN1-(4-Methoxy-phenyl)pseudo-UTP 19 N N1-(4-Amino-phenyl)pseudo-UTP 20 NN1(4-Nitro-phenyl)pseudo-UTP 21 N Pseudo-UTP-N1-p-benzoic acid 22 NN1-(4-Methyl-benzyl)pseudo-UTP 24 NN1-(2,4,6-Trimethyl-benzyl)pseudo-UTP 23 NN1-(4-Methoxy-benzyl)pseudo-UTP 25 N N1-(4-Amino-benzyl)pseudo-UTP 26 NN1-(4-Nitro-benzyl)pseudo-UTP 27 N Pseudo-UTP-N1-methyl-p-benzoic acid28 N N1-(2-Amino-ethyl)pseudo-UTP 29 N N1-(3-Amino-propyl)pseudo-UTP 30N N1-(4-Amino-butyl)pseudo-UTP 31 N N1-(5-Amino-pentyl)pseudo-UTP 32 NN1-(6-Amino-hexyl)pseudo-UTP 33 N Pseudo-UTP-N1-3-propionic acid 34 NPseudo-UTP-N1-4-butanoic acid 35 N Pseudo-UTP-N1-5-pentanoic acid 36 NPseudo-UTP-N1-6-hexanoic acid 37 N Pseudo-UTP-N1-7-heptanoic acid 38 NN1-(2-Amino-2-carboxyethyl)pseudo-UTP 39 NN1-(4-Amino-4-carboxybutyl)pseudo-UTP 40 N N3-Alkyl-pseudo-UTP 41 N6-Ethyl-pseudo-UTP 42 N 6-Propyl-pseudo-UTP 43 N 6-iso-Propyl-pseudo-UTP44 N 6-Butyl-pseudo-UTP 45 N 6-tert-Butyl-pseudo-UTP 46 N6-(2,2,2-Trifluoroethyl)-pseudo-UTP 47 N 6-Ethoxy-pseudo-UTP 48 N6-Trifluoromethoxy-pseudo-UTP 49 N 6-Phenyl-pseudo-UTP 50 N6-(Substituted-Phenyl)-pseudo-UTP 51 N 6-Cyano-pseudo-UTP 52 N6-Azido-pseudo-UTP 53 N 6-Amino-pseudo-UTP 54 N6-Ethylcarboxylate-pseudo-UTP  54b N 6-Hydroxy-pseudo-UTP 55 N6-Methylamino-pseudo-UTP  55b N 6-Dimethylamino-pseudo-UTP 57 N6-Hydroxyamino-pseudo-UTP 59 N 6-Formyl-pseudo-UTP 60 N6-(4-Morpholino)-pseudo-UTP 61 N 6-(4-Thiomorpholino)-pseudo-UTP 62 NN1-Me-4-thio-pseudo-UTP 63 N N1-Me-2-thio-pseudo-UTP 64 N1,6-Dimethyl-pseudo-UTP 65 N 1-Methyl-6-trifluoromethyl-pseudo-UTP 66 N1-Methyl-6-ethyl-pseudo-UTP 67 N 1-Methyl-6-propyl-pseudo-UTP 68 N1-Methyl-6-iso-propyl-pseudo-UTP 69 N 1-Methyl-6-butyl-pseudo-UTP 70 N1-Methyl-6-tert-butyl-pseudo-UTP 71 N1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP 72 N1-Methyl-6-iodo-pseudo-UTP 73 N 1-Methyl-6-bromo-pseudo-UTP 74 N1-Methyl-6-chloro-pseudo-UTP 75 N 1-Methyl-6-fluoro-pseudo-UTP 76 N1-Methyl-6-methoxy-pseudo-UTP 77 N 1-Methyl-6-ethoxy-pseudo-UTP 78 N1-Methyl-6-trifluoromethoxy-pseudo-UTP 79 N 1-Methyl-6-phenyl-pseudo-UTP80 N 1-Methyl-6-(substituted phenyl)pseudo-UTP 81 N1-Methyl-6-cyano-pseudo-UTP 82 N 1-Methyl-6-azido-pseudo-UTP 83 N1-Methyl-6-amino-pseudo-UTP 84 N 1-Methyl-6-ethylcarboxylate-pseudo-UTP85 N 1-Methyl-6-hydroxy-pseudo-UTP 86 N1-Methyl-6-methylamino-pseudo-UTP 87 N1-Methyl-6-dimethylamino-pseudo-UTP 88 N1-Methyl-6-hydroxyamino-pseudo-UTP 89 N 1-Methyl-6-formyl-pseudo-UTP 90N 1-Methyl-6-(4-morpholino)-pseudo-UTP 91 N1-Methyl-6-(4-thiomorpholino)-pseudo-UTP 92 N 1-Alkyl-6-vinyl-pseudo-UTP93 N 1-Alkyl-6-allyl-pseudo-UTP 94 N 1-Alkyl-6-homoallyl-pseudo-UTP 95 N1-Alkyl-6-ethynyl-pseudo-UTP 96 N 1-Alkyl-6-(2-propynyl)-pseudo-UTP 97 N1-Alkyl-6-(1-propynyl)-pseudo-UTP 98 N

Example 16 Incorporation of Naturally and Non-Naturally OccurringNucleosides

Naturally and non-naturally occurring nucleosides are incorporated intomRNA encoding a polypeptide of interest. Examples of these are given inTables 20 and 21. Certain commercially available nucleosidetriphosphates (NTPs) are investigated in the polynucleotides of theinvention. A selection of these are given in Table 20. The resultantmRNA are then examined for their ability to produce protein, inducecytokines, and/or produce a therapeutic outcome.

TABLE 20 Naturally and non-naturally occurring nucleosides NaturallyChemistry Modification Compound # occuring N4-Methyl-Cytosine 1 YN4,N4-Dimethyl-2′-OMe-Cytosine 2 Y 5-Oxyacetic acid-methyl ester-Uridine3 Y N3-Methyl-pseudo-Uridine 4 Y 5-Hydroxymethyl-Cytosine 5 Y5-Trifluoromethyl-Cytosine 6 N 5-Trifluoromethyl-Uridine 7 N5-Methyl-amino-methyl-Uridine 8 Y 5-Carboxy-methyl-amino-methyl-Uridine9 Y 5-Carboxymethylaminomethyl-2′-OMe-Uridine 10 Y5-Carboxymethylaminomethyl-2-thio-Uridine 11 Y5-Methylaminomethyl-2-thio-Uridine 12 Y5-Methoxy-carbonyl-methyl-Uridine 13 Y5-Methoxy-carbonyl-methyl-2′-OMe-Uridine 14 Y 5-Oxyacetic acid-Uridine15 Y 3-(3-Amino-3-carboxypropyl)-Uridine 16 Y5-(carboxyhydroxymethyl)uridine methyl ester 17 Y5-(carboxyhydroxymethyl)uridine 18 Y

TABLE 21 Non-naturally occurring nucleoside triphosphates NaturallyChemistry Modification Compound # occuring N1-Me-GTP 1 N2′-OMe-2-Amino-ATP 2 N 2′-OMe-pseudo-UTP 3 Y 2′-OMe-6-Me-UTP 4 N2′-Azido-2′-deoxy-ATP 5 N 2′-Azido-2′-deoxy-GTP 6 N2′-Azido-2′-deoxy-UTP 7 N 2′-Azido-2′-deoxy-CTP 8 N2′-Amino-2′-deoxy-ATP 9 N 2′-Amino-2′-deoxy-GTP 10 N2′-Amino-2′-deoxy-UTP 11 N 2′-Amino-2′-deoxy-CTP 12 N 2-Amino-ATP 13 N8-Aza-ATP 14 N Xanthosine-5′-TP 15 N 5-Bromo-CTP 16 N2′-F-5-Methyl-2′-deoxy-UTP 17 N 5-Aminoallyl-CTP 18 N2-Amino-riboside-TP 19 N

Example 17 Incorporation of Modifications to the Nucleobase andCarbohydrate (Sugar)

Naturally and non-naturally occurring nucleosides are incorporated intomRNA encoding a polypeptide of interest. Commercially availablenucleosides and NTPs having modifications to both the nucleobase andcarbohydrate (sugar) are examined for their ability to be incorporatedinto mRNA and to produce protein, induce cytokines, and/or produce atherapeutic outcome. Examples of these nucleosides are given in Tables22 and 23.

TABLE 22 Combination modifications Chemistry Modification Compound #5-iodo-2′-fluoro-deoxyuridine 1 5-iodo-cytidine 6 2′-bromo-deoxyuridine7 8-bromo-adenosine 8 8-bromo-guanosine 9 2,2′-anhydro-cytidinehydrochloride 10 2,2′-anhydro-uridine 11 2′-Azido-deoxyuridine 122-amino-adenosine 13 N4-Benzoyl-cytidine 14 N4-Amino-cytidine 152′-O-Methyl-N4-Acetyl-cytidine 16 2′Fluoro-N4-Acetyl-cytidine 172′Fluor-N4-Bz-cytidine 18 2′O-methyl-N4-Bz-cytidine 192′O-methyl-N6-Bz-deoxyadenosine 20 2′Fluoro-N6-Bz-deoxyadenosine 21N2-isobutyl-guanosine 22 2′Fluro-N2-isobutyl-guanosine 232′O-methyl-N2-isobutyl-guanosine 24

TABLE 23 Naturally occuring combinations Naturally Name Compound #occurring 5-Methoxycarbonylmethyl-2-thiouridine TP 1 Y5-Methylaminomethyl-2-thiouridine TP 2 Y 5-Crbamoylmethyluridine TP 3 Y5-Carbamoylmethyl-2′-O-methyluridine TP 4 Y1-Methyl-3-(3-amino-3-carboxypropyl) 5 Y pseudouridine TP5-Methylaminomethyl-2-selenouridine TP 6 Y 5-Carboxymethyluridine TP 7 Y5-Methyldihydrouridine TP 8 Y lysidine TP 9 Y 5-Taurinomethyluridine TP10 Y 5-Taurinomethyl-2-thiouridine TP 11 Y5-(iso-Pentenylaminomethyl)uridine TP 12 Y5-(iso-Pentenylaminomethyl)-2-thiouridine TP 13 Y5-(iso-Pentenylaminomethyl)-2′-O- 14 Y methyluridine TPN4-Acetyl-2′-O-methylcytidine TP 15 Y N4,2′-O-Dimethylcytidine TP 16 Y5-Formyl-2′-O-methylcytidine TP 17 Y 2′-O-Methylpseudouridine TP 18 Y2-Thio-2′-O-methyluridine TP 19 Y 3,2′-O-Dimethyluridine TP 20 Y

In the tables “UTP” stands for uridine triphosphate, “GTP” stands forguanosine triphosphate, “ATP” stands for adenosine triphosphate, “CTP”stands for cytosine triphosphate, “TP” stands for triphosphate and “Bz”stands for benzyl.

Example 18 Signal Sequence Exchange Study

Several variants of mmRNAs encoding human Granulocyte colony stimulatingfactor (G-CSF) (mRNA sequence shown in SEQ ID NO: 4258; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) weresynthesized using modified nucleotides pseudouridine and5-methylcytidine (pseudo-U/5mC). These variants included the G-CSFconstructs encoding either the wild-type N terminal secretory signalpeptide sequence (MAGPATQSPMKLMALQLLLWHSALWTVQEA; SEQ ID NO: 4271), nosecretory signal peptide sequence, or secretory signal peptide sequencestaken from other mRNAs. These included sequences where the wild typeGCSF signal peptide sequence was replaced with the signal peptidesequence of either: human a-1-anti trypsin (AAT)(MMPSSVSWGILLLAGLCCLVPVSLA; SEQ ID NO: 4272), human Factor IX (FIX)(MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKR; SEQ ID NO: 4273), humanProlactin (Prolac) (MKGSLLLLLVSNLLLCQSVAP; SEQ ID NO: 4274), or humanAlbumin (Alb) (MKWVTFISLLFLFSSAYSRGVFRR; SEQ ID NO: 4275).

250 ng of modified mRNA encoding each G-CSF variant was transfected intoHEK293A (293A in the table), mouse myoblast (MM in the table) (C2C12,CRL-1772, ATCC) and rat myoblast (RM in the table) (L6 line, CRL-1458,ATCC) cell lines in a 24 well plate using 1 ul of Lipofectamine 2000(Life Technologies), each well containing 300,000 cells. Thesupernatants were harvested after 24 hrs and the secreted G-CSF proteinwas analyzed by ELISA using the Human G-CSF ELISA kit (LifeTechnologies). The data shown in Table 24 reveal that cells transfectedwith G-CSF mmRNA encoding the Albumin signal peptide secrete at least 12fold more G-CSF protein than its wild type counterpart.

TABLE 24 Signal Peptide Exchange 293A MM RM Signal peptides (pg/ml)(pg/ml) (pg/ml) G-CSF Natural 9650 3450 6050 α-1-anti trypsin 9950 50008475 Factor IX 11675 6175 11675 Prolactin 7875 1525 9800 Albumin 12205081050 173300 No Signal peptide 0 0 0

Example 19 3′ Untranslated Regions

A 3′ UTR may be provided a flanking region. Multiple 3′ UTRs may beincluded in the flanking region and may be the same or of differentsequences. Any portion of the flanking regions, including none, may becodon optimized and any may independently contain one or more differentstructural or chemical modifications, before and/or after codonoptimization.

Shown in Table 7 is a listing of 3′-untranslated regions of theinvention. Variants of 3′ UTRs may be utilized wherein one or morenucleotides are added or removed to the termini, including A, T, C or G.

Example 20 Alteration of Polynucleotide Trafficking: NLS and NES

Two nuclear export signals (NES) which may be incorporated into thepolynucleotides of the present invention includes those reported byMuller, et al (Traffic, 2009, 10: 514-527) and are associated withsignaling via the gene COMMD1. These are NEST, PVAIIELEL (SEQ ID NO4276) and NES2, VNQILKTLSE (SEQ ID NO 4277).

Nuclear localization signals may also be used. One such sequence isPKKKRKV (SEQ ID NO: 4278).

Cell lines or mice are administered one or more polynucleotides having aNLS or NES encoded therein. Upon administration the polynucleotide istrafficked to an alternate location, e.g., into the nucleus using theNLS. The polypeptide having the NLS would be trafficked to the nucleuswhere it would deliver either a survival or death signal to the nuclearmicroenvironment. Polypeptides which may be localized to the nucleusinclude those with altered binding properties for DNA which willfunction to alter the expression profile of the cell in atherapeutically beneficial manner for the cell, tissue or organism.

In one experiment, the polynucleotide encodes a COMMD1 mut1/mut 2+NLS(e.g., both NES signals disrupted plus a NLS added) following themethods of Muller et al, (Traffic 2009; 10: 514-527) and van de Sluis etal, (J Clin Invest. 2010; 120 (6):2119-2130). The signal sequence mayencode a polypeptide or a scrambled sequence which is not translatable.The signal sequence encoded would interact with HIF1-alpha to alter thetranscritome of the cancer cells.

The experiment is repeated under normal and hypoxic conditions.

Once identified the HIF1-alpha dependent polynucleotide is tested incancer cell lines clonal survival or a marker of apoptosis is measuredand compared to control or mock treated cells.

Example 21 miRNA Binding Sites (BS) Useful as Sensor Sequences inPolynucleotides

miRNA-binding sites are used in the 3′UTR of mRNA therapeutics to directcytotoxic or cytoprotective mRNA therapeutics to specific cells (normaland/or cancerous).

A strong apoptotic signal (i.e., AIFsh—Apoptosis Inducing Factor shortisoform) is encoded as the polypeptide or “signal” and is encoded alongwith a series of 3′UTR miR binding sites, such as that for mir-122a,that would make the polynucleotide relatively much more stable incancerous cells than in normal cells.

Experiments comparing cancer vs. normal heaptic cell lines where thecancer cell lines have a specific miR signature are performed in vitro.SNU449 or HEP3B (human derived HCC cell lines) are used because bothhave been shown to have “undetectable miR-122a”, whereas normalhepatocytes should have very high miR-122a levels. First a cancer cellis selected which is sensitive to AIFsh polynucleotide (i.e., it resultsin apoptosis).

Three miR-122a binding sites are encoded into the 3′UTR of an mRNAsequence for AIFsh and the study arms include 2 cell lines (normalhepatocyte, SNU449 or HEP3B)×5 treatments (vehicle alone, polynucleotideuntranslatabe, polynucleotide AIFsh (no miR BS in 3′UTR), 3′UTR[miR122aBS×3]-polynucleotide untranslatable, 3′UTR[miR122a BS×3]-polynucleotideAIFsh).

The expected result would be significant apoptosis in the face ofpolynucleotide AIFsh in both normal and cancer (HEP3B or SNU449) celllines in the absence of any 3′UTR-miR122a BS. However, a significantdifference in the relative apoptosis of normal vs. cancer cell lines inthe face of 3′UTR [miR122a BS×3]-polynucleotide AIFsh.

Reversibility of the effect is shown with the co-administration ofmiR122a to the cancer cell line (e.g., through some transduction of themiR122a activity back into the cancer cell line).

In vivo animal studies are then performed using any of the modelsdisclosed herein or a commercially available orthotopic HCC model.

Example 22 Cell Lines for the Study of Polynucleotides

Polynucleotides of the present invention and formulations comprising thepolynucleotides of the present invention or described in Internationalapplication No PCT/US2012/69610, herein incorporated by reference in itsentirety, may be investigated in any number of cancer or normal celllines. Cell lines useful in the present invention include those fromATCC (Manassas, Va.) and are listed in Table 25.

TABLE 25 Cell lines ATCC Number Hybridoma or Cell line Description NameCCL-171 Homo sapiens (human) Source: Organ: lung MRC-5 Disease: normalCell Type: fibroblast CCL-185 Homo sapiens (human) Source: Organ: lungA549 Disease: carcinoma CCL-248 Homo sapiens (human) Source: Organ:colon T84 Disease: colorectal carcinoma Derived from metastatic site:lung CCL-256 Homo sapiens (human) Source: Organ: lung NCI-H2126 Disease:adenocarcinoma; non-small cell lung [H2126] cancer Derived frommetastatic site: pleural effusion CCL-257 Homo sapiens (human) Source:Organ: lung NCI-H1688 Disease: carcinoma; classic small cell lung[H1688] cancer CCL-75 Homo sapiens (human) Source: Organ: lung WI-38Disease: normal Cell Type: fibroblast CCL-75.1 Homo sapiens (human)Source: Organ: lung WI-38 VA-13 Cell Type: fibroblastSV40 transformedsubline 2RA CCL-95.1 Homo sapiens (human) Source: Organ: lung WI-26 VA4Cell Type: SV40 transformed CRL-10741 Homo sapiens (human) Source:Organ: liver C3A Disease: hepatocellular carcinoma [HepG2/C3A,derivative of Hep G2 (ATCC HB- 8065)] CRL-11233 Homo sapiens (human)Source: Organ: liver THLE-3 Tissue: left lobe Cell Type:epithelialimmortalized with SV40 large T antigen CRL-11351 Homo sapiens(human) Source: Organ: lung H69AR Disease: carcinoma; small cell lungcancer; multidrug resistant Cell Type: epithelial CRL-1848 Homo sapiens(human) Source: Organ: lung NCI-H292 [H292] Disease: mucoepidermoidpulmonary carcinoma CRL-1918 Homo sapiens (human) Source: Organ:pancreas CFPAC-1 Disease: ductal adenocarcinoma; cystic fibrosis Derivedfrom metastatic site: liver metastasis CRL-1973 Homo sapiens (human)Source: Organ: testis NTERA-2 cl.D1 Disease: malignant pluripotentembryonal [NT2/D1] carcinoma Derived from metastatic site: lung CRL-2049Homo sapiens (human) Source: Organ: lung DMS 79 Disease: carcinoma;small cell lung cancer CRL-2062 Homo sapiens (human) Source: Organ: lungDMS 53 Disease: carcinoma; small cell lung cancer CRL-2064 Homo sapiens(human) Source: Organ: lung DMS 153 Disease: carcinoma; small cell lungcancer Derived from metastatic site: liver CRL-2066 Homo sapiens (human)Source: Organ: lung DMS 114 Disease: carcinoma; small cell lung cancerCRL-2081 Homo sapiens (human) Source: Disease: biphasic MSTO-211Hmesothelioma Derived from metastatic site: lung CRL-2170 Homo sapiens(human) Source: Organ: lung SW 1573 [SW- Disease: alveolar cellcarcinoma 1573, SW1573] CRL-2177 Homo sapiens (human) Source: Organ:lung SW 1271 [SW- Disease: carcinoma; small cell lung cancer 1271,SW1271] CRL-2195 Homo sapiens (human) Source: Organ: lung SHP-77Disease: carcinoma; small cell lung cancer Cell Type: large cell,variant; CRL-2233 Homo sapiens (human) Source: Organ: liver SNU-398Disease: hepatocellular carcinoma CRL-2234 Homo sapiens (human) Source:Organ: liver SNU-449 Tumor Stage: grade II-III/IV Disease:hepatocellular carcinoma CRL-2235 Homo sapiens (human) Source: Organ:liver SNU-182 Tumor Stage: grade III/IV Disease: hepatocellularcarcinoma CRL-2236 Homo sapiens (human) Source: Organ: liver SNU-475Tumor Stage: grade II-IV/V Disease: hepatocellular carcinoma CRL-2237Homo sapiens (human) Source: Organ: liver SNU-387 Tumor Stage: gradeIV/V Disease: pleomorphic hepatocellular carcinoma CRL-2238 Homo sapiens(human) Source: Organ: liver SNU-423 Tumor Stage: grade III/IV Disease:pleomorphic hepatocellular carcinoma CRL-2503 Homo sapiens (human)Source: Organ: lung NL20 Tissue: bronchus Disease: normal CRL-2504 Homosapiens (human) Source: Organ: lung NL20-TA Tissue: bronchus [NL20T-A]Disease: normal CRL-2706 Homo sapiens (human) Source: Organ: liverTHLE-2 Tissue: left lobe Cell Type: epithelialSV40 transformed CRL-2741Homo sapiens (human) Source: Organ: lung HBE135-E6E7 Tissue: bronchusCell Type: epithelialHPV-16 E6/E7 transformed CRL-2868 Homo sapiens(human) Source: Organ: lung HCC827 Disease: adenocarcinoma Cell Type:epithelial CRL-2871 Homo sapiens (human) Source: Organ: lung HCC4006Disease: adenocarcinoma Derived from metastatic site: pleural effusionCell Type: epithelial CRL-5800 Homo sapiens (human) Source: Organ: lungNCI-H23 [H23] Disease: adenocarcinoma; non-small cell lung cancerCRL-5803 Homo sapiens (human) Source: Organ: lung NCI-H1299 Disease:carcinoma; non-small cell lung cancer Derived from metastatic site:lymph node CRL-5804 Homo sapiens (human) Source: Organ: lung NCI-H187[H187] Disease: carcinoma; classic small cell lung cancer Derived frommetastatic site: pleural effusion CRL-5807 Homo sapiens (human) Source:Organ: lung NCI-H358 [H- Tissue: bronchiole; alveolus 358, H358]Disease: bronchioalveolar carcinoma; non-small cell lung cancer CRL-5808Homo sapiens (human) Source: Organ: lung NCI-H378 [H378] Tumor Stage:stage E Disease: carcinoma; classic small cell lung cancer Derived frommetastatic site: pleural effusion CRL-5810 Homo sapiens (human) Source:Organ: lung NCI-H522 [H522] Tumor Stage: stage 2 Disease:adenocarcinoma; non-small cell lung cancer CRL-5811 Homo sapiens (human)Source: Organ: lung NCI-H526 [H526] Tumor Stage: stage E Disease:carcinoma; variant small cell lung cancer Derived from metastatic site:bone marrow CRL-5815 Homo sapiens (human) Source: Organ: lung NCI-H727[H727] Tissue: bronchus Disease: carcinoid CRL-5816 Homo sapiens (human)Source: Organ: lung NCI-H810 [H810] Tumor Stage: stage 2 Disease:carcinoma; non-small cell lung cancer CRL-5817 Homo sapiens (human)Source: Organ: lung NCI-H889 [H889] Tumor Stage: stage E Disease:carcinoma; classic small cell lung cancer Derived from metastatic site:lymph node CRL-5818 Homo sapiens (human) Source: Organ: lung NCI-H1155Disease: carcinoma; non-small cell lung cancer [H1155] Derived frommetastatic site: lymph node CRL-5819 Homo sapiens (human) Source: Organ:lung NCI-H1404 Disease: papillary adenocarcinoma [H1404] Derived frommetastatic site: lymph node CRL-5822 Homo sapiens (human) Source: Organ:stomach NCI-N87 [N87] Disease: gastric carcinoma Derived from metastaticsite: liver CRL-5823 Homo sapiens (human) Source: Organ: lung NCI-H196[H196] Tumor Stage: stage E Disease: carcinoma; variant small cell lungcancer Derived from metastatic site: pleural effusion CRL-5824 Homosapiens (human) Source: Organ: lung NCI-H211 [H211] Tumor Stage: stage EDisease: carcinoma; small cell lung cancer Derived from metastatic site:bone marrow CRL-5825 Homo sapiens (human) Source: Organ: lung NCI-H220[H220] Tumor Stage: stage E Disease: carcinoma; classic small cell lungcancer Derived from metastatic site: pleural effusion CRL-5828 Homosapiens (human) Source: Organ: lung NCI-H250 [H250] Tumor Stage: stage EDisease: carcinoma; classic small cell lung cancer Derived frommetastatic site: brain CRL-5831 Homo sapiens (human) Source: Organ: lungNCI-H524 [H524] Tumor Stage: stage L Disease: carcinoma; variant smallcell lung cancer Derived from metastatic site: lymph node CRL-5834 Homosapiens (human) Source: Organ: lung NCI-H647 [H647] Tumor Stage: stage3A Disease: adenosquamous carcinoma; non-small cell lung cancer Derivedfrom metastatic site: pleural effusion CRL-5835 Homo sapiens (human)Source: Organ: lung NCI-H650 [H650] Disease: bronchioalveolar carcinoma;non-small cell lung cancer Derived from metastatic site: lymph nodeCRL-5836 Homo sapiens (human) Source: Organ: lung NCI-H711 [H711] TumorStage: stage E Disease: carcinoma; classic small cell lung cancerDerived from metastatic site: bone marrow CRL-5837 Homo sapiens (human)Source: Organ: lung NCI-H719 [H719] Tumor Stage: stage E Disease:carcinoma; classic small cell lung cancer Derived from metastatic site:bone marrow CRL-5840 Homo sapiens (human) Source: Organ: lung NCI-H740[H740] Tumor Stage: stage E Disease: carcinoma; classic small cell lungcancer Derived from metastatic site: lymph node CRL-5841 Homo sapiens(human) Source: Organ: lung NCI-H748 [H748] Tumor Stage: stage EDisease: carcinoma; classic small cell lung cancer Derived frommetastatic site: lymph node CRL-5842 Homo sapiens (human) Source: Organ:lung NCI-H774 [H774] Tumor Stage: stage E Disease: carcinoma; classicsmall cell lung cancer Derived from metastatic site: soft tissueCRL-5844 Homo sapiens (human) Source: Organ: lung NCI-H838 [H838] Tumorstage: 3B Disease: adenocarcinoma; non-small cell lung cancer Derivedfrom metastatic site: lymph node CRL-5845 Homo sapiens (human) Source:Organ: lung NCI-H841 [H841] Tumor Stage: stage L Disease: carcinoma;variant small cell lung cancer Derived from metastatic site: lymph nodeCRL-5846 Homo sapiens (human) Source: Organ: lung NCI-H847 [H847] TumorStage: stage L Disease: carcinoma; classic small cell lung cancerDerived from metastatic site: pleural effusion CRL-5849 Homo sapiens(human) Source: Organ: lung NCI-H865 [H865] Tumor Stage: stage LDisease: carcinoma; classic small cell lung cancer Derived frommetastatic site: pleural effusion CRL-5850 Homo sapiens (human) Source:Organ: lung NCI-H920 [H920] Tumor Stage: stage 4 Disease:adenocarcinoma; non-small cell lung cancer Derived from metastatic site:lymph node CRL-5853 Homo sapiens (human) Source: Organ: lung NCI-H1048Disease: carcinoma; small cell lung cancer [H1048] Derived frommetastatic site: pleural effusion CRL-5855 Homo sapiens (human) Source:Organ: lung NCI-H1092 Tumor Stage: stage E [H1092] Disease: carcinoma;classic small cell lung cancer Derived from metastatic site: bone marrowCRL-5856 Homo sapiens (human) Source: Organ: lung NCI-H1105 Tumor Stage:stage E [H1105] Disease: carcinoma; classic small cell lung cancerDerived from metastatic site: lymph node CRL-5858 Homo sapiens (human)Source: Organ: lung NCI-H1184 Tumor Stage: stage L [H1184] Disease:carcinoma; small cell lung cancer Derived from metastatic site: lymphnode CRL-5859 Homo sapiens (human) Source: Organ: lung NCI-H1238 TumorStage: stage E [H1238] Disease: carcinoma; small cell lung cancerDerived from metastatic site: bone marrow CRL-5864 Homo sapiens (human)Source: Organ: lung NCI-H1341 Disease: carcinoma; small cell lung cancer[H1341] Derived from metastatic site: cervix CRL-5867 Homo sapiens(human) Source: Organ: lung NCI-H1385 Tumor Stage: stage 3A [H1385]Disease: carcinoma; non-small cell lung cancer Derived from metastaticsite: lymph node CRL-5869 Homo sapiens (human) Source: Organ: lungNCI-H1417 Tumor Stage: stage E [H1417] Disease: carcinoma; classic smallcell lung cancer CRL-5870 Homo sapiens (human) Source: Organ: lungNCI-H1435 Disease: adenocarcinoma; non-small cell lung [H1435] cancerCRL-5871 Homo sapiens (human) Source: Organ: lung NCI-H1436 Tumor Stage:stage E [H1436] Disease: carcinoma; classic small cell lung cancerDerived from metastatic site: lymph node CRL-5872 Homo sapiens (human)Source: Organ: lung NCI-H1437 Tumor Stage: stage 1 [H1437] Disease:adenocarcinoma; non-small cell lung cancer Derived from metastatic site:pleural effusion CRL-5874 Homo sapiens (human) Source: Organ: lungNCI-H1522 Tumor Stage: stage E [H1522] Disease: carcinoma; small celllung cancer Derived from metastatic site: pleural effusion CRL-5875 Homosapiens (human) Source: Organ: lung NCI-H1563 Disease: adenocarcinoma;non-small cell lung [H1563] cancer CRL-5876 Homo sapiens (human) Source:Organ: lung NCI-H1568 Disease: adenocarcinoma; non-small cell lung[H1568] cancer Derived from metastatic site: lymph node CRL-5877 Homosapiens (human) Source: Organ: lung NCI-H1573 Tumor Stage: stage 4[H1573] Disease: adenocarcinoma Derived from metastatic site: softtissue CRL-5878 Homo sapiens (human) Source: Organ: lung NCI-H1581 TumorStage: stage 4 [H1581] Disease: non-small cell lung cancer Cell Type:large cell; CRL-5879 Homo sapiens (human) Source: Tumor Stage: NCI-H1618stage E [H1618] Disease: carcinoma; small cell lung cancer Derived frommetastatic site: bone marrow CRL-5881 Homo sapiens (human) Source:Organ: lung NCI-H1623 Tumor Stage: stage 3B [H1623] Disease:adenocarcinoma; non-small cell lung cancer Derived from metastatic site:lymph node CRL-5883 Homo sapiens (human) Source: Organ: lung NCI-H1650[H- Tumor Stage: stage 3B 1650, H1650] Disease: adenocarcinoma;bronchoalveolar carcinoma Derived from metastatic site: pleural effusionCRL-5884 Homo sapiens (human) Source: Organ: lung NCI-H1651 Disease:adenocarcinoma; non-small cell lung [H1651] cancer CRL-5885 Homo sapiens(human) Source: Organ: lung NCI-H1666 [H- Disease: adenocarcinoma;bronchoalveolar 1666, H1666] carcinoma Derived from metastatic site:pleural effusion CRL-5886 Homo sapiens (human) Source: Organ: lungNCI-H1672 Tumor Stage: stage L [H1672] Disease: carcinoma; classic smallcell lung cancer CRL-5887 Homo sapiens (human) Source: Organ: lungNCI-H1693 Tumor Stage: stage 3B [H1693] Disease: adenocarcinoma;non-small cell lung cancer Derived from metastatic site: lymph nodeCRL-5888 Homo sapiens (human) Source: Organ: lung NCI-H1694 Tumor Stage:stage E [H1694] Disease: carcinoma; classic small cell lung cancerDerived from metastatic site: ascites CRL-5889 Homo sapiens (human)Source: Organ: lung NCI-H1703 Tumor Stage: stage 1 [H1703] Disease:non-small cell lung cancer Cell Type: squamous cell; CRL-5891 Homosapiens (human) Source: Organ: lung NCI-H1734 [H- Disease:adenocarcinoma; non-small cell lung 1734, H1734] cancer CRL-5892 Homosapiens (human) Source: Organ: lung NCI-H1755 Tumor Stage: stage 4[H1755] Disease: adenocarcinoma; non-small cell lung cancer Derived frommetastatic site: liver CRL-5892 Homo sapiens (human) Source: Organ: lungNCI-H1755 Tumor Stage: stage 4 [H1755] Disease: adenocarcinoma;non-small cell lung cancer Derived from metastatic site: liver CRL-5893Homo sapiens (human) Source: Organ: lung NCI-H1770 Tumor Stage: stage 4[H1770] Disease: carcinoma; non-small cell lung cancer Derived frommetastatic site: lymph node Cell Type: neuroendocrine; CRL-5896 Homosapiens (human) Source: Organ: lung NCI-H1793 Disease: adenocarcinoma;non-small cell lung [H1793] cancer CRL-5898 Homo sapiens (human) Source:Organ: lung NCI-H1836 Tumor Stage: stage L [H1836] Disease: carcinoma;classic small cell lung cancer CRL-5899 Homo sapiens (human) Source:Organ: lung NCI-H1838 Disease: adenocarcinoma; non-small cell lung[H1838] cancer CRL-5900 Homo sapiens (human) Source: Organ: lungNCI-H1869 Tumor Stage: stage 4 [H1869] Disease: non-small cell lungcancer Derived from metastatic site: pleural effusion Cell Type:squamous cell; CRL-5902 Homo sapiens (human) Source: Organ: lungNCI-H1876 Tumor Stage: stage E [H1876] Disease: carcinoma; classic smallcell lung cancer Derived from metastatic site: lymph node CRL-5903 Homosapiens (human) Source: Organ: lung NCI-H1882 Tumor Stage: stage E[H1882] Disease: carcinoma; small cell lung cancer Derived frommetastatic site: bone marrow CRL-5904 Homo sapiens (human) Source:Organ: lung NCI-H1915 Tumor Stage: stage 4 [H1915] Disease: poorlydifferentiated carcinoma; non- small cell lung cancer Derived frommetastatic site: brain Cell Type: large cell; CRL-5906 Homo sapiens(human) Source: Organ: lung NCI-H1930 Tumor Stage: stage L [H1930]Disease: carcinoma; classic small cell lung cancer Derived frommetastatic site: lymph node CRL-5907 Homo sapiens (human) Source: Organ:lung NCI-H1944 Tumor Stage: stage 3B [H1944] Disease: adenocarcinoma;non-small cell lung cancer Derived from metastatic site: soft tissueCRL-5908 Homo sapiens (human) Source: Organ: lung NCI-H1975 [H- Disease:adenocarcinoma; non-small cell lung 1975, H1975] cancer CRL-5909 Homosapiens (human) Source: Organ: lung NCI-H1993 Tumor Stage: stage 3A[H1993] Disease: adenocarcinoma; non-small cell lung cancer Derived frommetastatic site: lymph node CRL-5912 Homo sapiens (human) Source: Organ:lung NCI-H2023 Tumor Stage: stage 3A [H2023] Disease: adenocarcinoma;non-small cell lung cancer Derived from metastatic site: lymph nodeCRL-5913 Homo sapiens (human) Source: Organ: lung NCI-H2029 Tumor Stage:stage E [H2029] Disease: carcinoma; small cell lung cancer Derived frommetastatic site: lymph node CRL-5914 Homo sapiens (human) Source: Organ:lung NCI-H2030 Disease: adenocarcinoma; non-small cell lung [H2030]cancer Derived from metastatic site: lymph node CRL-5917 Homo sapiens(human) Source: Organ: lung NCI-H2066 Tumor Stage: stage 1 [H2066]Disease: mixed; small cell lung cancer; adenocarcinoma; squamous cellcarcinoma CRL-5918 Homo sapiens (human) Source: Organ: lung NCI-H2073Tumor Stage: stage 3A [H2073] Disease: adenocarcinoma; non-small celllung cancer CRL-5920 Homo sapiens (human) Source: Organ: lung NCI-H2081Tumor Stage: stage E [H2081] Disease: carcinoma; classic small cell lungcancer Derived from metastatic site: pleural effusion CRL-5921 Homosapiens (human) Source: Organ: lung NCI-H2085 Disease: adenocarcinoma;non-small cell lung [H2085] cancer CRL-5922 Homo sapiens (human) Source:Organ: lung NCI-H2087 Tumor Stage: stage 1 [H2087] Disease:adenocarcinoma; non-small cell lung cancer Derived from metastatic site:lymph node CRL-5923 Homo sapiens (human) Source: Organ: lung NCI-H2106Tissue: neuroendocrine [H2106] Tumor Stage: stage 4 Disease: non-smallcell lung cancer Derived from metastatic site: lymph node CRL-5924 Homosapiens (human) Source: Organ: lung NCI-H2110 Disease: non-small celllung cancer [H2110] Derived from metastatic site: pleural effusionCRL-5926 Homo sapiens (human) Source: Organ: lung NCI-H2135 Disease:non-small cell lung cancer [H2135] CRL-5927 Homo sapiens (human) Source:Organ: lung NCI-H2141 Tumor Stage: stage E [H2141] Disease: carcinoma;small cell lung cancer Derived from metastatic site: lymph node CRL-5929Homo sapiens (human) Source: Organ: lung NCI-H2171 Tumor Stage: stage E[H2171] Disease: carcinoma; small cell lung cancer Derived frommetastatic site: pleural effusion CRL-5930 Homo sapiens (human) Source:Organ: lung NCI-H2172 Disease: non-small cell lung cancer [H2172]CRL-5931 Homo sapiens (human) Source: Organ: lung NCI-H2195 Tumor Stage:stage E [H2195] Disease: carcinoma; small cell lung cancer Derived frommetastatic site: bone marrow CRL-5932 Homo sapiens (human) Source:Organ: lung NCI-H2196 Tumor Stage: stage E [H2196] Disease: carcinoma;small cell lung cancer Derived from metastatic site: bone marrowCRL-5933 Homo sapiens (human) Source: Organ: lung NCI-H2198 Tumor Stage:stage E [H2198] Disease: carcinoma; small cell lung cancer Derived frommetastatic site: lymph node CRL-5934 Homo sapiens (human) Source: Organ:lung NCI-H2227 Tumor Stage: stage E [H2227] Disease: carcinoma; smallcell lung cancer CRL-5935 Homo sapiens (human) Source: Organ: lungNCI-H2228 Disease: adenocarcinoma; non-small cell lung [H2228] cancerCRL-5938 Homo sapiens (human) Source: Organ: lung NCI-H2286 Tumor Stage:stage 1 [H2286] Disease: mixed; small cell lung cancer; adenocarcinoma;squamous cell carcinoma CRL-5939 Homo sapiens (human) Source: Organ:lung NCI-H2291 Disease: adenocarcinoma; non-small cell lung [H2291]cancer Derived from metastatic site: lymph node CRL-5940 Homo sapiens(human) Source: Organ: lung NCI-H2330 Tumor Stage: stage L [H2330]Disease: carcinoma; small cell lung cancer Derived from metastatic site:lymph node CRL-5941 Homo sapiens (human) Source: Organ: lung NCI-H2342Tumor Stage: stage 3A [H2342] Disease: adenocarcinoma; non-small celllung cancer CRL-5942 Homo sapiens (human) Source: Organ: lung NCI-H2347Tumor Stage: stage 1 [H2347] Disease: adenocarcinoma; non-small celllung cancer CRL-5944 Homo sapiens (human) Source: Organ: lung NCI-H2405Tumor Stage: stage 4 [H2405] Disease: adenocarcinoma; non-small celllung cancer Derived from metastatic site: ascites CRL-5945 Homo sapiens(human) Source: Organ: lung NCI-H2444 Disease: non-small cell lungcancer [H2444] CRL-5975 Homo sapiens (human) Source: Organ: lung UMC-11Disease: carcinoid CRL-5976 Homo sapiens (human) Source: Organ: lungNCI-H64 [H64] Tumor Stage: stage E Disease: carcinoma; small cell lungcancer Derived from metastatic site: lymph node CRL-5978 Homo sapiens(human) Source: Organ: lung NCI-H735 [H735] Tumor Stage: stage EDisease: carcinoma; small cell lung cancer Derived from metastatic site:liver CRL-5978 Homo sapiens (human) Source: Organ: lung NCI-H735 [H735]Tumor Stage: stage E Disease: carcinoma; small cell lung cancer Derivedfrom metastatic site: liver CRL-5982 Homo sapiens (human) Source: Organ:lung NCI-H1963 Tumor Stage: stage L [H1963] Disease: carcinoma; smallcell lung cancer CRL-5983 Homo sapiens (human) Source: Organ: lungNCI-H2107 Tumor Stage: stage E [H2107] Disease: carcinoma; small celllung cancer Derived from metastatic site: bone marrow CRL-5984 Homosapiens (human) Source: Organ: lung NCI-H2108 Tumor Stage: stage E[H2108] Disease: carcinoma; small cell lung cancer Derived frommetastatic site: bone marrow CRL-5985 Homo sapiens (human) Source:Organ: lung NCI-H2122 Tumor Stage: stage 4 [H2122] Disease:adenocarcinoma; non-small cell lung cancer Derived from metastatic site:pleural effusion CRL-7343 Homo sapiens (human) Source: Organ: lung Hs573.T Disease: cancer CRL-7344 Homo sapiens (human) Source: Organ: lungHs 573.Lu CRL-8024 Homo sapiens (human) Source: Organ: liver PLC/PRF/5Disease: hepatoma Cell Type: Alexander cells; CRL-9609 Homo sapiens(human) Source: Organ: lung BEAS-2B Tissue: bronchus Disease: normalCell Type: epithelialvirus transformed HB-8065 Homo sapiens (human)Source: Organ: liver Hep G2 Disease: hepatocellular carcinoma HTB-105Homo sapiens (human) Source: Organ: testes Tera-1 Disease: embryonalcarcinoma, malignant Derived from metastatic site: lung HTB-106 Homosapiens (human) Source: Disease: Tera-2 malignant embryonal carcinomaDerived from metastatic site: lung HTB-119 Homo sapiens (human) Source:Organ: lung NCI-H69 [H69] Disease: carcinoma; small cell lung cancerHTB-120 Homo sapiens (human) Source: Organ: lung NCI-H128 [H128]Disease: carcinoma; small cell lung cancer Derived from metastatic site:pleural effusion HTB-168 Homo sapiens (human) Source: Organ: lungChaGo-K-1 Tissue: bronchus Disease: bronchogenic carcinoma HTB-171 Homosapiens (human) Source: Organ: lung NCI-H446 [H446] Disease: carcinoma;small cell lung cancer Derived from metastatic site: pleural effusionHTB-172 Homo sapiens (human) Source: Organ: lung NCI-H209 [H209]Disease: carcinoma; small cell lung cancer Derived from metastatic site:bone marrow HTB-173 Homo sapiens (human) Source: Organ: lung NCI-H146[H146] Disease: carcinoma; small cell lung cancer Derived frommetastatic site: bone marrow HTB-174 Homo sapiens (human) Source: Organ:lung NCI-H441 [H441] Disease: papillary adenocarcinoma HTB-175 Homosapiens (human) Source: Organ: lung NCI-H82 [H82] Disease: carcinoma;small cell lung cancer Derived from metastatic site: pleural effusionHTB-177 Homo sapiens (human) Source: Organ: lung NCI-H460 [H460]Disease: carcinoma; large cell lung cancer Derived from metastatic site:pleural effusion HTB-178 Homo sapiens (human) Source: Organ: lungNCI-H596 [H596] Disease: adenosquamous carcinoma HTB-179 Homo sapiens(human) Source: Organ: lung NCI-H676B Disease: adenocarcinoma [H676B]Derived from metastatic site: pleural effusion HTB-180 Homo sapiens(human) Source: Organ: lung NCI-H345 [H345] Disease: carcinoma; smallcell lung cancer Derived from metastatic site: bone marrow HTB-181 Homosapiens (human) Source: Organ: lung NCI-H820 [H820] Disease: papillaryadenocarcinoma Derived from metastatic site: lymph node HTB-182 Homosapiens (human) Source: Organ: lung NCI-H520 [H520] Disease: squamouscell carcinoma HTB-183 Homo sapiens (human) Source: Organ: lung NCI-H661[H661] Disease: carcinoma; large cell lung cancer Derived frommetastatic site: lymph node HTB-184 Homo sapiens (human) Source: Organ:lung NCI-H510A Disease: carcinoma; small cell lung cancer; [H510A, NCI-extrapulmonary origin H510] Derived from metastatic site: adrenal glandHTB-52 Homo sapiens (human) Source: Organ: liver SK-HEP-1 Tissue:ascites Disease: adenocarcinoma HTB-53 Homo sapiens (human) Source:Organ: lung A-427 Disease: carcinoma HTB-54 Homo sapiens (human) Source:Organ: lung Calu-1 Tumor Stage: grade III Disease: epidermoid carcinomaDerived from metastatic site: pleura HTB-55 Homo sapiens (human) Source:Organ: lung Calu-3 Disease: adenocarcinoma Derived from metastatic site:pleural effusion HTB-56 Homo sapiens (human) Source: Organ: unknown,Calu-6 probably lung Disease: anaplastic carcinoma HTB-57 Homo sapiens(human) Source: Organ: lung SK-LU-1 Disease: adenocarcinoma HTB-58 Homosapiens (human) Source: Organ: lung SK-MES-1 Disease: squamous cellcarcinoma Derived from metastatic site: pleural effusion HTB-59 Homosapiens (human) Source: Organ: lung SW 900 [SW-900, Tumor Stage: gradeIV SW900] Disease: squamous cell carcinoma HTB-64 Homo sapiens (human)Source: Disease: Malme-3M malignant melanoma Derived from metastaticsite: lung HTB-79 Homo sapiens (human) Source: Organ: pancreas Capan-1Disease: adenocarcinoma Derived from metastatic site: liver

Example 23 RNA Binding Proteins

RNA binding proteins may be provided as proteins and/or as nucleic acidsencoding such proteins. RNA binding proteins play a multitude of rolesin regulating RNA stability and protein translation. In someembodiments, RNA binding proteins are provided in protein and/or nucleicacid form with elements of the present invention. Such RNA bindingproteins include, but are not limited to those listed (along with theENSG number, identifying the corresponding gene as well as one or moreENST number, identifying transcriptional variants of each) in Table 26.

TABLE 26 RNA binding proteins Protein ENST SEQ ENSP SEQ No. RNA bindingprotein ENSG ENST ID NO ENSP ID NO 1 AU RNA binding protein/enoyl-148090 422391 4279 402026 4632 CoA hydratase 2 AU RNA bindingprotein/enoyl- 148090 303617 4280 307334 4633 CoA hydratase 3 AU RNAbinding protein/enoyl- 148090 375731 4281 364883 4634 CoA hydratase 4cold inducible RNA binding 99622 320936 4282 322887 4635 protein 5 coldinducible RNA binding 99622 444172 4283 407512 4636 protein 6 coldinducible RNA binding 99622 413636 4284 412831 4637 protein 7 cold shockdomain containing 172346 306149 4285 302485 4638 C2, RNA binding 8heterogeneous nuclear 138668 543098 4286 439380 4639 ribonucleoprotein D(AU-rich element RNA binding protein 1, 37 kDa) 9 heterogeneous nuclear138668 313899 4287 313199 4640 ribonucleoprotein D (AU-rich element RNAbinding protein 1, 37 kDa) 10 heterogeneous nuclear 138668 541060 4288437416 4641 ribonucleoprotein D (AU-rich element RNA binding protein 1,37 kDa) 11 heterogeneous nuclear 138668 503822 4289 422615 4642ribonucleoprotein D (AU-rich element RNA binding protein 1, 37 kDa) 12heterogeneous nuclear 138668 507010 4290 421952 4643 ribonucleoprotein D(AU-rich element RNA binding protein 1, 37 kDa) 13 heterogeneous nuclear138668 353341 4291 313327 4644 ribonucleoprotein D (AU-rich element RNAbinding protein 1, 37 kDa) 14 heterogeneous nuclear 138668 514671 4292426446 4645 ribonucleoprotein D (AU-rich element RNA binding protein 1,37 kDa) 15 heterogeneous nuclear 138668 352301 4293 305860 4646ribonucleoprotein D (AU-rich element RNA binding protein 1, 37 kDa) 16heterogeneous nuclear 138668 307213 4294 307544 4647 ribonucleoprotein D(AU-rich element RNA binding protein 1, 37 kDa) 17 insulin-like growthfactor 2 159217 290341 4295 290341 4648 mRNA binding protein 1 18insulin-like growth factor 2 73792 382199 4296 371634 4649 mRNA bindingprotein 2 19 insulin-like growth factor 2 73792 421047 4297 413787 4650mRNA binding protein 2 20 insulin-like growth factor 2 73792 346192 4298320204 4651 mRNA binding protein 2 21 insulin-like growth factor 2136231 258729 4299 258729 4652 mRNA binding protein 3 22 KH domaincontaining, RNA 121774 327300 4300 313829 4653 binding, signaltransduction associated 1 23 KH domain containing, RNA 121774 4929894301 417731 4654 binding, signal transduction associated 1 24 KH domaincontaining, RNA 121774 355201 4302 347336 4655 binding, signaltransduction associated 1 25 KH domain containing, RNA 112232 2811564303 281156 4656 binding, signal transduction associated 2 26 KH domaincontaining, RNA 112232 539571 4304 443437 4657 binding, signaltransduction associated 2 27 KH domain containing, RNA 131773 3558494305 348108 4658 binding, signal transduction associated 3 28 QKI, KHdomain containing, 112531 361752 4306 355094 4659 RNA binding 29 QKI, KHdomain containing, 112531 275262 4307 275262 4660 RNA binding 30 QKI, KHdomain containing, 112531 392127 4308 375973 4661 RNA binding 31 QKI, KHdomain containing, 112531 361195 4309 354867 4662 RNA binding 32 QKI, KHdomain containing, 112531 453779 4310 408775 4663 RNA binding 33 RALYRNA binding protein-like 184672 522613 4311 427787 4664 34 RALY RNAbinding protein-like 184672 523850 4312 428807 4665 35 RALY RNA bindingprotein-like 184672 521695 4313 428667 4666 36 RALY RNA bindingprotein-like 184672 521268 4314 430367 4667 37 RALY RNA bindingprotein-like 184672 517988 4315 428711 4668 38 RALY RNA bindingprotein-like 184672 522455 4316 430394 4669 39 RD RNA binding protein204356 375425 4317 364574 4670 40 RD RNA binding protein 204356 4448114318 388400 4671 41 RD RNA binding protein 204356 441998 4319 3979144672 42 RD RNA binding protein 204356 375429 4320 364578 4673 43 RD RNAbinding protein 204356 426722 4321 394340 4674 44 RD RNA binding protein204356 454913 4322 409389 4675 45 RD RNA binding protein 206268 4117454323 410872 4676 46 RD RNA binding protein 206268 456281 4324 3969714677 47 RD RNA binding protein 206268 440478 4325 407528 4678 48 RD RNAbinding protein 206268 383174 4326 372660 4679 49 RD RNA binding protein206268 551833 4327 447903 4680 50 RD RNA binding protein 206268 4586224328 409139 4681 51 RD RNA binding protein 206357 548056 4329 4498974682 52 RD RNA binding protein 206357 434518 4330 409269 4683 53 RD RNAbinding protein 206357 449057 4331 393793 4684 54 RD RNA binding protein206357 383343 4332 372834 4685 55 RD RNA binding protein 206357 4200394333 411487 4686 56 RD RNA binding protein 206357 425810 4334 4036304687 57 RD RNA binding protein 229363 549252 4335 450250 4688 58 RD RNAbinding protein 229363 448628 4336 394879 4689 59 RD RNA binding protein229363 424762 4337 415567 4690 60 RD RNA binding protein 229363 4184234338 395175 4691 61 RD RNA binding protein 229363 418059 4339 4013424692 62 RD RNA binding protein 229363 453084 4340 393794 4693 63 RD RNAbinding protein 231044 443464 4341 393103 4694 64 RD RNA binding protein231044 548988 4342 449910 4695 65 RD RNA binding protein 231044 4298574343 403623 4696 66 RD RNA binding protein 231044 437732 4344 3975654697 67 RD RNA binding protein 231044 424967 4345 411724 4698 68 RD RNAbinding protein 231044 420837 4346 414014 4699 69 RD RNA binding protein233801 456263 4347 407630 4700 70 RD RNA binding protein 233801 4521474348 401745 4701 71 RD RNA binding protein 233801 457397 4349 3930054702 72 RD RNA binding protein 233801 552869 4350 447844 4703 73 RD RNAbinding protein 233801 425721 4351 390689 4704 74 RD RNA binding protein233801 435435 4352 396604 4705 75 RNA binding motif (RNP1, 102317 4303484353 412764 4706 RRM) protein 3 76 RNA binding motif (RNP1, 102317376759 4354 365950 4707 RRM) protein 3 77 RNA binding motif (RNP1,102317 376755 4355 365946 4708 RRM) protein 3 78 RNA binding motif(RNP1, 102317 354480 4356 346473 4709 RRM) protein 3 79 RNA bindingmotif protein 10 182872 377604 4357 366829 4710 80 RNA binding motifprotein 10 182872 329236 4358 328848 4711 81 RNA binding motif protein10 182872 345781 4359 329659 4712 82 RNA binding motif protein 11 185272400577 4360 383421 4713 83 RNA binding motif protein 12 244462 3596464361 352668 4714 84 RNA binding motif protein 12 244462 374104 4362363217 4715 85 RNA binding motif protein 12 244462 374114 4363 3632284716 86 RNA binding motif protein 12 244462 431148 4364 392642 4717 87RNA binding motif protein 12 244462 424458 4365 411036 4718 88 RNAbinding motif protein 12 244462 435161 4366 411692 4719 89 RNA bindingmotif protein 12 244462 349942 4367 339879 4720 90 RNA binding motifprotein 12B 183808 518597 4368 428269 4721 91 RNA binding motif protein12B 183808 399300 4369 382239 4722 92 RNA binding motif protein 12B183808 520560 4370 429807 4723 93 RNA binding motif protein 12B 183808521947 4371 430466 4724 94 RNA binding motif protein 12B 183808 5177004372 427729 4725 95 RNA binding motif protein 12B 183808 519109 4373430474 4726 96 RNA binding motif protein 14 239306 310137 4374 3117474727 97 RNA binding motif protein 15 162775 369784 4375 358799 4728 98RNA binding motif protein 15B 179837 323686 4376 313890 4729 99 RNAbinding motif protein 15B 179837 536338 4377 444388 4730 100 RNA bindingmotif protein 15B 179837 541145 4378 443941 4731 101 RNA binding motifprotein 15B 179837 540284 4379 437933 4732 102 RNA binding motif protein17 134453 447032 4380 406024 4733 103 RNA binding motif protein 17134453 437845 4381 395448 4734 104 RNA binding motif protein 17 134453372795 4382 361881 4735 105 RNA binding motif protein 17 134453 4186314383 402303 4736 106 RNA binding motif protein 17 134453 432931 4384408214 4737 107 RNA binding motif protein 17 134453 379888 4385 3692184738 108 RNA binding motif protein 17 134453 446108 4386 388638 4739 109RNA binding motif protein 18 119446 417201 4387 409315 4740 110 RNAbinding motif protein 19 122965 545145 4388 442053 4741 111 RNA bindingmotif protein 19 122965 261741 4389 261741 4742 112 RNA binding motifprotein 19 122965 392561 4390 376344 4743 113 RNA binding motif protein20 203867 539821 4391 446400 4744 114 RNA binding motif protein 20203867 369519 4392 358532 4745 115 RNA binding motif protein 22 86589447771 4393 412118 4746 116 RNA binding motif protein 22 86589 1998144394 199814 4747 117 RNA binding motif protein 22 86589 540000 4395441594 4748 118 RNA binding motif protein 23 100461 399922 4396 3828064749 119 RNA binding motif protein 23 100461 359890 4397 352956 4750 120RNA binding motif protein 23 100461 346528 4398 339220 4751 121 RNAbinding motif protein 23 100461 554618 4399 451448 4752 122 RNA bindingmotif protein 23 100461 553876 4400 450672 4753 123 RNA binding motifprotein 23 100461 557571 4401 452382 4754 124 RNA binding motif protein23 100461 557549 4402 450558 4755 125 RNA binding motif protein 23100461 338980 4403 345496 4756 126 RNA binding motif protein 23 100461554256 4404 452583 4757 127 RNA binding motif protein 23 100461 5574644405 451403 4758 128 RNA binding motif protein 23 100461 555691 4406452538 4759 129 RNA binding motif protein 23 100461 556862 4407 4525574760 130 RNA binding motif protein 23 100461 555676 4408 451364 4761 131RNA binding motif protein 24 112183 379052 4409 368341 4762 132 RNAbinding motif protein 24 112183 318204 4410 319551 4763 133 RNA bindingmotif protein 24 112183 425446 4411 396898 4764 134 RNA binding motifprotein 25 119707 525161 4412 434004 4765 135 RNA binding motif protein25 119707 525321 4413 436868 4766 136 RNA binding motif protein 25119707 531500 4414 434333 4767 137 RNA binding motif protein 25 119707261973 4415 261973 4768 138 RNA binding motif protein 25 119707 5274324416 431150 4769 139 RNA binding motif protein 25 119707 526754 4417436225 4770 140 RNA binding motif protein 25 119707 540173 4418 4379344771 141 RNA binding motif protein 26 139746 267229 4419 267229 4772 142RNA binding motif protein 26 139746 327303 4420 327080 4773 143 RNAbinding motif protein 26 139746 438724 4421 390222 4774 144 RNA bindingmotif protein 27 91009 265271 4422 265271 4775 145 RNA binding motifprotein 28 106344 223073 4423 223073 4776 146 RNA binding motif protein33 184863 438356 4424 405793 4777 147 RNA binding motif protein 33184863 287912 4425 287912 4778 148 RNA binding motif protein 33 184863401878 4426 384160 4779 149 RNA binding motif protein 33 184863 3411484427 341583 4780 150 RNA binding motif protein 34 188739 408888 4428386226 4781 151 RNA binding motif protein 34 188739 400947 4429 3837314782 152 RNA binding motif protein 34 188739 429912 4430 413409 4783 153RNA binding motif protein 34 188739 366606 4431 355565 4784 154 RNAbinding motif protein 38 132819 356208 4432 348538 4785 155 RNA bindingmotif protein 38 132819 440234 4433 407848 4786 156 RNA binding motifprotein 38 132819 371219 4434 360263 4787 157 RNA binding motif protein39 131051 253363 4435 253363 4788 158 RNA binding motif protein 39131051 407261 4436 384541 4789 159 RNA binding motif protein 39 131051361162 4437 354437 4790 160 RNA binding motif protein 39 131051 4483034438 394824 4791 161 RNA binding motif protein 39 131051 374038 4439363150 4792 162 RNA binding motif protein 39 131051 528062 4440 4367474793 163 RNA binding motif protein 39 131051 338163 4441 344581 4794 164RNA binding motif protein 39 131051 434927 4442 393493 4795 165 RNAbinding motif protein 4 173933 532968 4443 432020 4796 166 RNA bindingmotif protein 4 173933 408993 4444 386561 4797 167 RNA binding motifprotein 4 173933 409406 4445 386894 4798 168 RNA binding motif protein 4173933 483858 4446 435821 4799 169 RNA binding motif protein 4 173933310092 4447 309166 4800 170 RNA binding motif protein 41 89682 4348544448 405522 4801 171 RNA binding motif protein 41 89682 372479 4449361557 4802 172 RNA binding motif protein 41 89682 372487 4450 3615654803 173 RNA binding motif protein 41 89682 372482 4451 361560 4804 174RNA binding motif protein 41 89682 203616 4452 203616 4805 175 RNAbinding motif protein 42 126254 262633 4453 262633 4806 176 RNA bindingmotif protein 42 126254 360475 4454 353663 4807 177 RNA binding motifprotein 43 184898 331426 4455 331211 4808 178 RNA binding motif protein44 177483 316997 4456 321179 4809 179 RNA binding motif protein 44177483 409864 4457 386727 4810 180 RNA binding motif protein 45 155636286070 4458 286070 4811 181 RNA binding motif protein 45 155636 4559034459 415940 4812 182 RNA binding motif protein 46 151962 281722 4460281722 4813 183 RNA binding motif protein 47 163694 295971 4461 2959714814 184 RNA binding motif protein 47 163694 381793 4462 371212 4815 185RNA binding motif protein 47 163694 511902 4463 425111 4816 186 RNAbinding motif protein 47 163694 515053 4464 422564 4817 187 RNA bindingmotif protein 47 163694 511598 4465 424019 4818 188 RNA binding motifprotein 47 163694 513473 4466 421589 4819 189 RNA binding motif protein47 163694 505414 4467 423527 4820 190 RNA binding motif protein 47163694 514782 4468 426542 4821 191 RNA binding motif protein 47 163694319592 4469 320108 4822 192 RNA binding motif protein 47 163694 5071804470 423398 4823 193 RNA binding motif protein 47 163694 381795 4471371214 4824 194 RNA binding motif protein 47 163694 505220 4472 4255074825 195 RNA binding motif protein 48 127993 509224 4473 442073 4826 196RNA binding motif protein 48 127993 450580 4474 401920 4827 197 RNAbinding motif protein 48 127993 265732 4475 265732 4828 198 RNA bindingmotif protein 4B 173914 525754 4476 433071 4829 199 RNA binding motifprotein 4B 173914 310046 4477 310471 4830 200 RNA binding motif protein5 3756 469838 4478 419534 4831 201 RNA binding motif protein 5 3756347869 4479 343054 4832 202 RNA binding motif protein 5 3756 441305 4480390711 4833 203 RNA binding motif protein 5 3756 543047 4481 442591 4834204 RNA binding motif protein 5 3756 536082 4482 445347 4835 205 RNAbinding motif protein 5 3756 417905 4483 406119 4836 206 RNA bindingmotif protein 5 3756 437500 4484 394622 4837 207 RNA binding motifprotein 5 3756 544851 4485 439808 4838 208 RNA binding motif protein 53756 539538 4486 440744 4839 209 RNA binding motif protein 6 4534 4229554487 392939 4840 210 RNA binding motif protein 6 4534 442092 4488 3935304841 211 RNA binding motif protein 6 4534 425608 4489 408665 4842 212RNA binding motif protein 6 4534 443081 4490 396466 4843 213 RNA bindingmotif protein 6 4534 416583 4491 390202 4844 214 RNA binding motifprotein 6 4534 433811 4492 389763 4845 215 RNA binding motif protein 64534 539992 4493 443165 4846 216 RNA binding motif protein 6 4534 2660224494 266022 4847 217 RNA binding motif protein 7 76053 540163 4495439918 4848 218 RNA binding motif protein 8A 131795 369307 4496 3583134849 219 RNA binding motif protein 8A 131795 330165 4497 333001 4850 220RNA binding motif protein, X- 147274 449161 4498 415250 4851 linked 221RNA binding motif protein, X- 147274 320676 4499 359645 4852 linked 222RNA binding motif protein, X- 147274 419968 4500 405117 4853 linked 223RNA binding motif protein, X- 147274 431446 4501 411989 4854 linked 224RNA binding motif protein, X- 134597 305536 4502 339090 4855 linked 2225 RNA binding motif protein, X- 134597 370947 4503 359985 4856 linked2 226 RNA binding motif protein, X- 134597 538614 4504 437425 4857linked 2 227 RNA binding motif protein, X- 213516 399794 4505 4460994858 linked-like 1 228 RNA binding motif protein, X- 213516 321792 4506318415 4859 linked-like 1 229 RNA binding motif protein, X- 170748306904 4507 304139 4860 linked-like 2 230 RNA binding motif protein, X-175718 424776 4508 417451 4861 linked-like 3 231 RNA binding motifprotein, Y- 234414 382707 4509 372154 4862 linked, family 1, member A1232 RNA binding motif protein, Y- 234414 439108 4510 388006 4863 linked,family 1, member A1 233 RNA binding motif protein, Y- 234414 303902 4511303712 4864 linked, family 1, member A1 234 RNA binding motif protein,Y- 242875 383020 4512 372484 4865 linked, family 1, member B 235 RNAbinding motif protein, Y- 244395 418956 4513 399181 4866 linked, family1, member D 236 RNA binding motif protein, Y- 244395 382680 4514 3721274867 linked, family 1, member D 237 RNA binding motif protein, Y- 244395382677 4515 372124 4868 linked, family 1, member D 238 RNA binding motifprotein, Y- 242389 382658 4516 372104 4869 linked, family 1, member E239 RNA binding motif protein, Y- 242389 382659 4517 372105 4870 linked,family 1, member E 240 RNA binding motif protein, Y- 242389 382673 4518372119 4871 linked, family 1, member E 241 RNA binding motif protein, Y-169800 303766 4519 307155 4872 linked, family 1, member F 242 RNAbinding motif protein, Y- 169800 454978 4520 406005 4873 linked, family1, member F 243 RNA binding motif protein, Y- 226941 414629 4521 4057454874 linked, family 1, member J 244 RNA binding motif protein, Y- 226941250831 4522 250831 4875 linked, family 1, member J 245 RNA binding motifprotein, Y- 226941 445779 4523 389621 4876 linked, family 1, member J246 RNA binding motif, single 153250 348849 4524 294904 4877 strandedinteracting protein 1 247 RNA binding motif, single 153250 428519 4525389016 4878 stranded interacting protein 1 248 RNA binding motif, single153250 409075 4526 386347 4879 stranded interacting protein 1 249 RNAbinding motif, single 153250 409972 4527 387280 4880 strandedinteracting protein 1 250 RNA binding motif, single 153250 392753 4528376508 4881 stranded interacting protein 1 251 RNA binding motif, single153250 409289 4529 386571 4882 stranded interacting protein 1 252 RNAbinding motif, single 76067 262031 4530 262031 4883 stranded interactingprotein 2 253 RNA binding motif, single 144642 434693 4531 395592 4884stranded interacting protein 3 254 RNA binding motif, single 144642383767 4532 373277 4885 stranded interacting protein 3 255 RNA bindingmotif, single 144642 383766 4533 373276 4886 stranded interactingprotein 3 256 RNA binding motif, single 144642 456853 4534 400519 4887stranded interacting protein 3 257 RNA binding motif, single 144642396583 4535 379828 4888 stranded interacting protein 3 258 RNA bindingmotif, single 144642 273139 4536 273139 4889 stranded interactingprotein 3 259 RNA binding protein S1, serine- 205937 320225 4537 3158594890 rich domain 260 RNA binding protein S1, serine- 205937 301730 4538301730 4891 rich domain 261 RNA binding protein S1, serine- 205937397086 4539 380275 4892 rich domain 262 RNA binding protein with 157110320203 4540 318102 4893 multiple splicing 263 RNA binding protein with157110 287771 4541 287771 4894 multiple splicing 264 RNA binding proteinwith 157110 339877 4542 340176 4895 multiple splicing 265 RNA bindingprotein with 157110 538486 4543 445406 4896 multiple splicing 266 RNAbinding protein with 157110 397323 4544 380486 4897 multiple splicing267 RNA binding protein with 166831 300069 4545 300069 4898 multiplesplicing 2 268 RNA binding protein, 125970 246194 4546 246194 4899autoantigenic (hnRNP-associated with lethal yellow homolog (mouse)) 269RNA binding protein, 125970 442805 4547 415973 4900 autoantigenic(hnRNP-associated with lethal yellow homolog (mouse)) 270 RNA bindingprotein, 125970 448364 4548 413638 4901 autoantigenic (hnRNP-associatedwith lethal yellow homolog (mouse)) 271 RNA binding protein, 125970413297 4549 403744 4902 autoantigenic (hnRNP-associated with lethalyellow homolog (mouse)) 272 RNA binding protein, 125970 375114 4550364255 4903 autoantigenic (hnRNP-associated with lethal yellow homolog(mouse)) 273 RNA binding protein, fox-1 78328 311745 4551 309117 4904homolog (C. elegans) 1 274 RNA binding protein, fox-1 78328 550418 4552450031 4905 homolog (C. elegans) 1 275 RNA binding protein, fox-1 78328355637 4553 347855 4906 homolog (C. elegans) 1 276 RNA binding protein,fox-1 78328 553186 4554 447753 4907 homolog (C. elegans) 1 277 RNAbinding protein, fox-1 78328 436368 4555 402745 4908 homolog (C.elegans) 1 278 RNA binding protein, fox-1 78328 352951 4556 322925 4909homolog (C. elegans) 1 279 RNA binding protein, fox-1 78328 340209 4557344196 4910 homolog (C. elegans) 1 280 RNA binding protein, fox-1 78328547338 4558 447717 4911 homolog (C. elegans) 1 281 RNA binding protein,fox-1 78328 547372 4559 446842 4912 homolog (C. elegans) 1 282 RNAbinding protein, fox-1 78328 551752 4560 447281 4913 homolog (C.elegans) 1 283 RNA binding protein, fox-1 100320 397303 4561 380470 4914homolog (C. elegans) 2 284 RNA binding protein, fox-1 100320 397305 4562380472 4915 homolog (C. elegans) 2 285 RNA binding protein, fox-1 100320338644 4563 342831 4916 homolog (C. elegans) 2 286 RNA binding protein,fox-1 100320 408983 4564 386177 4917 homolog (C. elegans) 2 287 RNAbinding protein, fox-1 100320 438146 4565 413035 4918 homolog (C.elegans) 2 288 RNA binding protein, fox-1 100320 359369 4566 352328 4919homolog (C. elegans) 2 289 RNA binding protein, fox-1 100320 262829 4567262829 4920 homolog (C. elegans) 2 290 RNA binding protein, fox-1 100320405409 4568 384944 4921 homolog (C. elegans) 2 291 RNA binding protein,fox-1 100320 449924 4569 391670 4922 homolog (C. elegans) 2 292 RNAbinding protein, fox-1 100320 414461 4570 407855 4923 homolog (C.elegans) 2 293 RNA binding protein, fox-1 100320 416721 4571 405651 4924homolog (C. elegans) 2 294 RNA binding protein, fox-1 167281 415831 4572408395 4925 homolog (C. elegans) 3 295 RNA binding protein, fox-1 167281453134 4573 393262 4926 homolog (C. elegans) 3 296 S1 RNA binding domain1 68784 535761 4574 263736 4927 297 S1 RNA binding domain 1 68784 2637364575 441272 4928 298 SERPINE1 mRNA binding 142864 370995 4576 3600344929 protein 1 299 SERPINE1 mRNA binding 142864 361219 4577 354591 4930protein 1 300 SERPINE1 mRNA binding 142864 370994 4578 360033 4931protein 1 301 SERPINE1 mRNA binding 142864 370990 4579 360029 4932protein 1 302 signal recognition particle 14 kDa 140319 267884 4580267884 4933 (homologous Alu RNA binding protein) 303 spermatidperinuclear RNA 165209 407982 4581 384292 4934 binding protein 304spermatid perinuclear RNA 165209 348403 4582 321347 4935 binding protein305 spermatid perinuclear RNA 165209 479114 4583 431531 4936 bindingprotein 306 spermatid perinuclear RNA 165209 447404 4584 415968 4937binding protein 307 spermatid perinuclear RNA 165209 360998 4585 3542714938 binding protein 308 SRA stem-loop interacting RNA 119705 5573424586 450909 4939 binding protein 309 staufen, RNA binding protein,124214 371856 4587 360922 4940 homolog 1 (Drosophila) 310 staufen, RNAbinding protein, 124214 360426 4588 353604 4941 homolog 1 (Drosophila)311 staufen, RNA binding protein, 124214 371828 4589 360893 4942 homolog1 (Drosophila) 312 staufen, RNA binding protein, 124214 371805 4590360870 4943 homolog 1 (Drosophila) 313 staufen, RNA binding protein,124214 371802 4591 360867 4944 homolog 1 (Drosophila) 314 staufen, RNAbinding protein, 124214 456866 4592 398785 4945 homolog 1 (Drosophila)315 staufen, RNA binding protein, 124214 347458 4593 323443 4946 homolog1 (Drosophila) 316 staufen, RNA binding protein, 124214 340954 4594345425 4947 homolog 1 (Drosophila) 317 staufen, RNA binding protein,124214 371792 4595 360857 4948 homolog 1 (Drosophila) 318 staufen, RNAbinding protein, 124214 437404 4596 416779 4949 homolog 1 (Drosophila)319 staufen, RNA binding protein, 40341 355780 4597 348026 4950 homolog2 (Drosophila) 320 staufen, RNA binding protein, 40341 524300 4598428756 4951 homolog 2 (Drosophila) 321 staufen, RNA binding protein,40341 524104 4599 430611 4952 homolog 2 (Drosophila) 322 staufen, RNAbinding protein, 40341 522695 4600 428456 4953 homolog 2 (Drosophila)323 staufen, RNA binding protein, 40341 522509 4601 427977 4954 homolog2 (Drosophila) 324 staufen, RNA binding protein, 40341 521736 4602428737 4955 homolog 2 (Drosophila) 325 staufen, RNA binding protein,40341 521447 4603 428829 4956 homolog 2 (Drosophila) 326 TAR (HIV-1) RNAbinding 59588 40877 4604 40877 4957 protein 1 327 TAR (HIV-1) RNAbinding 139546 456234 4605 416077 4958 protein 2 328 TAR (HIV-1) RNAbinding 139546 266987 4606 266987 4959 protein 2 329 TIA1 cytotoxicgranule- 116001 433529 4607 401371 4960 associated RNA binding protein330 TIA1 cytotoxic granule- 116001 477807 4608 445092 4961 associatedRNA binding protein 331 TIA1 cytotoxic granule- 116001 415783 4609404023 4962 associated RNA binding protein 332 TIA1 cytotoxic granule-151923 412524 4610 403573 4963 associated RNA binding protein- like 1333 TIA1 cytotoxic granule- 151923 436547 4611 394902 4964 associatedRNA binding protein- like 1 334 TIA1 cytotoxic granule- 151923 3690864612 358082 4965 associated RNA binding protein- like 1 335 TIA1cytotoxic granule- 151923 369093 4613 358089 4966 associated RNA bindingprotein- like 1 336 TIA1 cytotoxic granule- 151923 369092 4614 3580884967 associated RNA binding protein- like 1 337 zinc finger CCHC-typeand RNA 139168 266529 4615 266529 4968 binding motif 1 338 zinc fingerRNA binding protein 56097 265069 4616 265069 4969 339 zinc finger RNAbinding protein 56097 416900 4617 393243 4970 340 zinc finger RNAbinding protein 56097 382126 4618 371560 4971 341 zinc finger RNAbinding protein 2 105278 439086 4619 388567 4972 342 zinc finger RNAbinding protein 2 105278 262961 4620 262961 4973 343 zinc finger RNAbinding protein 2 105278 438164 4621 388974 4974 344 cold shock domaincontaining 9307 261443 4622 261443 4975 E1, RNA-binding 345 cold shockdomain containing 9307 339438 4623 342408 4976 E1, RNA-binding 346 coldshock domain containing 9307 358528 4624 351329 4977 E1, RNA-binding 347cold shock domain containing 9307 369530 4625 358543 4978 E1,RNA-binding 348 cold shock domain containing 9307 438362 4626 4077244979 E1, RNA-binding 349 cold shock domain containing 9307 525878 4627431562 4980 E1, RNA-binding 350 cold shock domain containing 9307 5259704628 432805 4981 E1, RNA-binding 351 cold shock domain containing 9307530886 4629 431297 4982 E1, RNA-binding 352 cold shock domain containing9307 534389 4630 435185 4983 E1, RNA-binding 353 cold shock domaincontaining 9307 534699 4631 432958 4984 E1, RNA-binding

HuR is a stabilizing AREBP. To increase the stability of the mRNA ofinterest, an mRNA encoding HuR can be co-transfected or co-injectedalong with the mRNA of interest into the cells or into the tissue. Theseproteins can also be tethered to the mRNA of interest in vitro and thenadministered to the cells together. Poly A tail binding protein, PABPinteracts with eukaryotic translation initiation factor eIF4G tostimulate translational initiation. Co-administration of mRNAs encodingthese RBPs along with the mRNA drug and/or tethering these proteins tothe mRNA drug in vitro and administering the protein-bound mRNA into thecells can increase the translational efficiency of the mRNA. The sameconcept can be extended to co-administration of mRNA along with mRNAsencoding various translation factors and facilitators as well as withthe proteins themselves to influence RNA stability and/or translationalefficiency.

Example 24 Expression of Modified Nucleic Acid with microRNA BindingSite

Human embryonic kidney epithelial cells (HEK293A), antigen presentingcells or cell lines with highly expressed mir-142/146, such asmonocyte-derived dendritic cells (MDDC) or PBMC, are seeded at a densityof 200,000 per well in 500 ul cell culture medium (InVitro GRO mediumfrom Celsis, Chicago, Ill.). G-CSF mRNA (mRNA sequence is shown in SEQID NO: 4258; polyA tail of at least 140 nucleotides not shown insequence; 5′Cap, Cap1) G-CSF mRNA having a miR-142-5p binding site(G-CSF miR-142-5p) (cDNA sequence is shown in SEQ ID NO: 4985; mRNAsequence is shown in SEQ ID NO: 4986, polyA tail of at least 140nucleotides not shown in sequence; 5′Cap, Cap1), G-CSF mRNA having aseed sequence from miR-142-5p binding site (G-CSF miR-142-5p-seed) (cDNAsequence is shown in SEQ ID NO: 4987; mRNA sequence is shown in SEQ IDNO: 4988; polyA tail of at least 140 nucleotides not shown in sequence;5′Cap, Cap1) G-CSF mRNA having a miR-142-5p binding site without theseed sequence (G-CSF miR-142-5p-seedless) (cDNA sequence is shown in SEQID NO: 4989, mRNA sequence is shown in SEQ ID NO: 4990; polyA tail of atleast 140 nucleotides not shown in sequence; 5′Cap, Cap1) G-CSF mRNAhaving a miR-142-3p binding site (G-CSF miR-142-3p) (cDNA sequence isshown in SEQ ID NO: 4991; mRNA sequence is shown in SEQ ID NO: 4992;polyA tail of at least 140 nucleotides not shown in sequence; 5′Cap,Cap1) G-CSF mRNA having a seed sequence from miR-142-3p binding site(G-CSF miR-142-3p-seed) (cDNA sequence is shown in SEQ ID NO: 4993; mRNAsequence is shown in SEQ ID NO: 4994; polyA tail of at least 140nucleotides not shown in sequence; 5′Cap, Cap1) G-CSF mRNA having amiR-142-3p binding site without the seed sequence (G-CSFmiR-142-3p-seedless) (DNA sequence is shown in SEQ ID NO: 4995; mRNAsequence is shown in SEQ ID NO: 4996; polyA tail of at least 140nucleotides not shown in sequence; 5′Cap, Cap1) G-CSF mRNA having amiR-146a binding site (G-CSF miR-146a) (cDNA sequence is shown in SEQ IDNO: 4997; mRNA sequence is shown in SEQ ID NO: 4998; polyA tail of atleast 140 nucleotides not shown in sequence; 5′Cap, Cap1) G-CSF mRNAhaving a seed sequence from miR-146a binding site (G-CSF miR-146a-seed)(cDNA sequence is shown in SEQ ID NO:4999; mRNA sequence is shown in SEQID NO: 5000; polyA tail at least 140 nucleotides not shown in sequence;5′Cap, Cap1) or G-CSF mRNA having a miR-146a binding site without theseed sequence (G-CSF miR-146a-seedless) (cDNA sequence is shown in SEQID NO: 5001; mRNA sequence is shown in SEQ ID NO: 5002; polyA tail atleast nucleotides not shown in sequence; 5′Cap, Cap1) are tested at aconcentration of 250 ng per well in 24 well plates. The mRNA sequencesare evaluated with various chemical modifications described hereinand/or known in the art including, fully modified with 5-methylcytidineand pseudouridine, fully modified with 5-methylcytidine and1-methylpseudouridine, fully modified with pseudouridine, fully modifiedwith 1-methylpseudouridine and where 25% of the uridine residues aremodified with 2-thiouridine and 25% of the cytosine residues aremodified with 5-methylcytidine. The expression of G-CSF in each sampleis measured by ELISA.

Shown in Table 27 are the DNA and mRNA G-CSF sequences with the miRbinding sites described above. In the table, the start codon of eachsequence is underlined.

TABLE 27 G-CSF Constructs with miR binding sites SEQ ID NO. DescriptionSEQ 4985 DNA TAATACGACTCACTATA sequenceGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAG having theCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTAT T7GGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCC polymeraseAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCA site andTTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCG restrictionATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACT sites:TTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGG G-CSF miR-ATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA 142-5pGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTAGTAGTGCTTTCTACTTTATGTGGTCTTTGAATAAAGCCTGAGTAGGAAG GCGGCCGCTCGAGCATGCATCTAGA4986 mRNA GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA sequence:GCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUU G-CSF miR-AUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACA 142-5pGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUAGUAGUGCUUUCUACUUUAUGUG GUCUUUGAAUAAAGCCUGAGUAGGAAG4987 DNA TAATACGACTCACTATA sequenceGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAG having theCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTAT T7GGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCC polymeraseAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCA site andTTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCG restrictionATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACT sites:TTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGG G-CSF miR-ATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA 142-5p-seedGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTACTTTATTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGA GCATGCATCTAGA 4988 mRNAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA sequence: GCCACC G-CSF miRAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCC 142-5p-seedCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGG GUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUACUUUAUUGGU CUUUGAAUAAAGCCUGAGUAGGAAG4989 DNA TAATACGACTCACTATA sequenceGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAG having theCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTAT T7GGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCC polymeraseAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCA site andTTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCG restrictionATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACT sites:TTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGG G-CSF miR-ATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA 142-5p-GTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGT seedlessATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTAGTAGTGCTTTCTGTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCC GCTCGAGCATGCATCTAGA 4990mRNA GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA sequence: GCCACCG-CSF miR- AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCC 142-5p-CUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAA seedlessGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGG GUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUAGUAGUGCUUUCUGUGGUCUUUGAAUAAAGCCUGAGUAGGAAG 4991 DNA TAATACGACTCACTATA sequenceGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAG having theCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTAT T7GGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCC polymeraseAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCA site andTTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCG restrictionATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACT sites:TTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGG G-CSF miR-ATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA 142-3pGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTCCATAAAGTAGGAAACACTACATGGTCTTTGAATAAAGCCTGAGTAGG AAGGCGGCCGCTCGAGCATGCATCTAGA4992 mRNA GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA sequence: GCCACCG-CSF miR AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCC 142-3pCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGG GUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUCCAUAAAGUAGGAAACACUACAUGGUCUUUGAAUAAAGCCUGAGUAGGAAG 4993 DNA TAATACGACTCACTATAsequence GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAG having theCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTAT T7GGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCC polymeraseAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCA site andTTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCG restrictionATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACT sites:TTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGG G-CSF miR-ATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA 142-3p-seedGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTACACTACTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGA GCATGCATCTAGA 4994 mRNAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA sequence: GCCACC G-CSF miR-AUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCC 142-3p-seedCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGG GUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUACACUACUGGU CUUUGAAUAAAGCCUGAGUAGGAAG4995 DNA TAATACGACTCACTATA sequenceGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAG having theCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTAT T7GGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCC polymeraseAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCA site andTTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCG restrictionATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACT sites:TTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGG G-CSF miR-ATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA 142-3p-GTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGT seedlessATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTCCATAAAGTAGGAAATGGTCTTTGAATAAAGCCTGAGTAGGAAGGCG GCCGCTCGAGCATGCATCTAGA 4996mRNA GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA sequence:GCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUU G-CSF miR-AUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACA 142-3p-GUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCG seedlessCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUCCAUAAAGUAGGAAAUGGUCUU UGAAUAAAGCCUGAGUAGGAAG 4997DNA TAATACGACTCACTATA sequenceGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAG having theCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTAT T7GGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCC polymeraseAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCA site andTTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCG restrictionATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACT sites:TTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGG G-CSF miR-ATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA 146aGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTAACCCATGGAATTCAGTTCTCATGGTCTTTGAATAAAGCCTGAGTAGGAA GGCGGCCGCTCGAGCATGCATCTAGA4998 mRNA GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA sequence:GCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUU G-CSF miR-AUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACA 146aGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUAACCCAUGGAAUUCAGUUCUCAU GGUCUUUGAAUAAAGCCUGAGUAGGAAG4999 DNA TAATACGACTCACTATA sequenceGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAG having theCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTAT T7GGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCC polymeraseAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCA site andTTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCG restrictionATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACT sites:TTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGG G-CSF-ATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA 146a-seedGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTAGTTCTCTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGA GCATGCATCTAGA 5000 mRNAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA sequence:GCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUU G-CSF-AUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACA 146a-seedGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUAGUUCUCUGGUCUUUGAAUAAAG CCUGAGUAGGAAG 5001 DNATAATACGACTCACTATA sequence GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGhaving the CCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTAT T7GGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCC polymeraseAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCA site andTTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCG restrictionATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACT sites:TTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGG G-CSF-ATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCA 146a-GTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGT seedlessATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTAACCCATGGAATTCATGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGC CGCTCGAGCATGCATCTAGA 5002mRNA GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA sequence:GCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUU G-CSF-AUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACA 146a-GUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCG seedlessCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUAACCCAUGGAAUUCAUGGUCUUU GAAUAAAGCCUGAGUAGGAAG

It is like that the binding site “seed” sequence is sufficient to inducemicroRNA binding, the expression of G-CSF should be down-regulated incells transfected with miR-142-3p, miR-142-3p-seed, miR-142-5p,miR-142-5p-seed, miR-146a or miR-146a-seed. Whereas, themiR-142-3p-seedless, miR-142-5p-seedless, miR-146a-seedless should notchange the expression of G-CSF, as compared with cells transfected withG-CSF mRNA without microRNA binding sites.

Example 25 APCs Specific microRNA Binding Sites to Suppress ModifiedNucleic Acid Mediated Immune Stimulation

The binding sites for microRNAs are used in the 3′UTR of mRNAtherapeutics to selectively degrade mRNA therapeutics in the immunecells to subdue unwanted immunogenic reactions caused by mRNAtherapeutics delivery.

A polynucleotide comprising a series of 3′UTR miR binding sites whichmake the nucleic acids or mRNA of the present invention more unstable inantigen presenting cells (APCs), such as, but not limited to mir-142-5p,mir-142-3p, mir-146a-5p and mir-146a-3p, encodes an oncology-relatedpolypeptide of the present invention. The addition of miR binding sitesin the 3′UTR making a signal sensor polynucleotide unstable would subduemodified mRNA mediated immune stimulation.

Experiments comparing the cytokine expression (e.g. TNF-alpha) inducedby the polypeptide with APCs specific microRNA signature vs. withoutsuch signature is performed in vitro by methods described herein and/orknown in the art.

Example 26 In Vitro Expression of mRNAs with miR Binding Sites

Human embryonic kidney epithelial cells (HEK293A), antigen-presentingcells or cell lines with highly expressed mir-142/146, such asmonocyte-derived dendritic cells (MDDC) or PBMC, are seeded at a densityof 200,000 per well in 500 ul cell culture medium (InVitro GRO mediumfrom Celsis, Chicago, Ill.). Cultured cells are transfected with G-CSFmRNAs with or without microRNA signature, as described in Example 24.The cells are transfected for five consecutive days. The transfectioncomplexes are removed four hours after each round of transfection.

The culture supernatant is assayed for secreted G-CSF (R&D Systems,catalog #DCS50), tumor necrosis factor-alpha (TNF-alpha) and interferonalpha (IFN-alpha by ELISA every day after transfection followingmanufacturer's protocols. The cells are analyzed for viability usingCELL TITER GLO® (Promega, catalog #G7570) 6 hrs and 18 hrs after thefirst round of transfection and every alternate day following that. Atthe same time from the harvested cells, total RNA is isolated andtreated with DNASE®using the RNAEASY micro kit (catalog #74004)following the manufacturer's protocol. 100 ng of total RNA is used forcDNA synthesis using the High Capacity cDNA Reverse Transcription kit(Applied Biosystems, cat #4368814) following the manufacturer'sprotocol. The cDNA is then analyzed for the expression of innate immuneresponse genes by quantitative real time PCR using SybrGreen in a BioradCFX 384 instrument following the manufacturer's protocol.

Example 27 In Vivo Detection of Innate Immune Response Study

To test the nucleic acids or mRNA protein expression and in vivo immuneresponse, female BALB/C mice (n=5) are injected intramuscularly withG-CSF mRNA with or without microRNA signatures as described in Example24. Blood is collected at 8 hours after dosing. The protein levels ofG-CSF, TNF-alpha and IFN-alpha is determined by ELISA.

The difference of cytokine production is seen as measured by mouseTNF-alpha and IFN-alpha level in serum. Injection with G-CSF modifiedmRNA having miR-142 and miR-146a binding site or binding site seed showsa lower level of cytokine response in vivo.

Example 28 Expression of miR-122 in Primary Hepatocytes

Hepatocyte specific miR-122 level in rat and human primary hepatocyteswas measured. Hela Cells and primary rat and human hepatocytes werecultured and RNAs were extracted from cell lysates. The miR-122 level inrat and human primary hepatocytes was compared with that in Hela cells.The miR-122 level is about 6 fold increased in primary human hepatocytesand about 12 fold increased in primary rat hepatocytes, respectively, ascompared with that in Hela cells.

Example 29 Expression of Modified Nucleic Acid with mir-122 Binding Sitein Hepatocytes

Primary rat and human hepatocytes and Hela cells were seeded at adensity of 200,000 per well in 500 ul cell culture medium (InVitro GROmedium from Celsis, Chicago, Ill.). G-CSF mRNA having a miR-122 bindingsite in the 3′UTR (G-CSF miR-122-1X) (mRNA sequence is shown in SEQ IDNO: 4268; polyA tail of approximately 140 nucleotides not shown insequence; 5′Cap, Cap1) fully modified with 5-methylcytidine andpseudouridine (5mC/pU), or fully modified with pseudouridine (pU) orG-CSF mRNA with four miR-122 binding sites with the seed deleted (G-CSFno seed) (mRNA sequence is shown in SEQ ID NO: 4270; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′Cap, Cap1) fullymodified with 5-methylcytidine and pseudouridine (5mC/pU) or fullymodified with pseudouridine (pU) was tested at a concentration of 250 ngper well in 24 well plates. The 24 hours after transfection, theexpression of G-CSF was measured by ELISA, and the results are shown inTable 28.

TABLE 28 G-CSF mir122 expression Primary human Primary rat Hela cellsHepatocytes Hepatocytes Protein Protein Protein Expression ExpressionExpression (ng/mL) (ng/mL) (ng/mL) G-CSF miR-122 167.34 67.60 3.40 1X(5mC/pU) G-CSF miR-122 292.18 116.18 25.63 1X (pU) G-CSF no seed 194.78129.77 8.39 (5mC/pU) G-CSF no seed 335.78 462.88 84.93 (pU)

Example 30 Expression of Modified Nucleic Acids with mir-122 BindingSites in Hepatocytes

MicroRNA control gene expression through the translational suppressionand/or degradation of target messenger RNA. Mir-122 binding sitecontaining G-CSF mRNA was translationally regulated in hepatocytes.

Primary rat and human hepatocytes and Hela cells were seeded at adensity of 200,000 per well in 500 ul cell culture medium (InVitro GROmedium from Celsis, Chicago, Ill.). G-CSF mRNA (G-CSF alpha) (mRNAsequence is shown in SEQ ID NO: 4266; polyA tail of approximately 140nucleotides not shown in sequence; 5′Cap, Cap1) fully modified with5-methylcytidine and pseudouridine (5mC/pU), G-CSF mRNA having a miR-122binding site in the 3′UTR (G-CSF miR-122-1X) (mRNA sequence is shown inSEQ ID NO: 4268; polyA tail of approximately 140 nucleotides not shownin sequence; 5′Cap, Cap 1) fully modified with 5-methylcytidine andpseudouridine (5mc/pU) or G-CSF mRNA with four miR-122 binding siteswith the seed deleted (G-CSF no seed) (mRNA sequence is shown in SEQ IDNO: 4270; polyA tail of approximately 140 nucleotides not shown insequence; 5′Cap, Cap1) fully modified with 5-methylcytidine andpseudouridine (5mC/pU) was tested at a concentration of 250 ng per wellin 24 well plates. 24 hours after transfection, the expression of G-CSFwas measured by ELISA. The G-CSF drug (mRNA) levels and protein levelsare shown in Table 29.

TABLE 29 G-CSF drug and protein levels Human Hepatocytes Drug RatHepatocytes (mRNA) Drug (mRNA) level level (unit Protein (unit Proteinnormalized expression normalized to expression to HPRT) (ng/ml) HPRT)(ng/ml) G-CSF alpha 43237.6 247.26 26615.88 784.6 (5mC/pU) G-CSF miR-46340.9 74.07 20171.07 40.628 122-1X (5mC/pU) G-CSF no seed 70239.7298.28 23170.47 894.06 (5mC/pU)

Example 31 Modified mRNA Sequences with or without Kozak and/or IRESSequences

Modified mRNA encoding G-CSF with or without a Kozak and/or IRESsequence, and their corresponding cDNA sequences, are shown below inTable 30. In Table 30, the start codon of each sequence is underlined.

TABLE 30 G-CSF Sequences SEQ ID Sequence NO: G-CSFOptimized G-CSF cDNA sequence containing a T7 polymerase site, 5003 withkozak sequence, IRES and Xba1 restriction site: Kozak TAATACGACTCACTATAsequence GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG and IRES AGCCACC andTCGTGAGGATCTATTTCCGGTGAATTCCTCGAGACTAGTTCT humanAGAGCGGCCGCGGATCCCGCCCCTCTCCCTCCCCCCCCCCTA alpha-ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCG globinTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCA 3′UTRATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCACCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAA ACACGATGATAATATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTCTAGA mRNA sequence (transcribed): 5004GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCUCGUGAGGAUCUAUUUCCGGUGAAUUCCUCGAGACUAGUUCUAGAGCGGCCGCGGAUCCCGCCCCUCUCCCUCCCCCCCCCCUAACGUUACUGGCCGAAGCCGCUUGGAAUAAGGCCGGUGUGCGUUUGUCUAUAUGUUAUUUUCCACCAUAUUGCCGUCUUUUGGCAAUGUGAGGGCCCGGAAACCUGGCCCUGUCUUCUUGACGAGCAUUCCUAGGGGUCUUUCCCCUCUCGCCAAAGGAAUGCAAGGUCUGUUGAAUGUCGUGAAGGAAGCAGUUCCUCUGGAAGCUUCUUGAAGACAAACAACGUCUGUAGCGACCCUUUGCAGGCAGCGGAACCCCCCACCUGGCGACAGGUGCCUCUGCGGCCAAAAGCCACGUGUAUAAGAUACACCUGCAAAGGCGGCACAACCCCAGUGCCACGUUGUGAGUUGGAUAGUUGUGGAAAGAGUCAAAUGGCUCACCUCAAGCGUAUUCAACAAGGGGCUGAAGGAUGCCCAGAAGGUACCCCAUUGUAUGGGAUCUGAUCUGGGGCCUCGGUGCACAUGCUUUACAUGUGUUUAGUCGAGGUUAAAAAACGUCUAGGCCCCCCGAACCACGGGGACGUGGUUUUCCUUUGAAAAACACGAUGAUAAUAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACA UCUUGCGCAGCCG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGU GGUCUUUGAAUAAAGUCUGAGUGGGCGGCG-CSF Optimized G-CSF cDNA sequence containing a T7 polymerase site,5005 without a IRES and Xba1 restriction site: Kozak TAATACGACTCACTATAsequence GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA and withTCGTGAGGATCTATTTCCGGTGAATTCCTCGAGACTAGTTCT an IRESAGAGCGGCCGCGGATCCCGCCCCTCTCCCTCCCCCCCCCCTA andACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCG humanTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCA alpha-ATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGC globinATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGT 3′UTRCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCACCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAA ACACGATGATAATATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTCTAGA mRNA sequence (transcribed): 5006GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAUCGUGAGGAUCUAUUUCCGGUGAAUUCCUCGAGACUAGUUCUAGAGCGGCCGCGGAUCCCGCCCCUCUCCCUCCCCCCCCCCUAACGUUACUGGCCGAAGCCGCUUGGAAUAAGGCCGGUGUGCGUUUGUCUAUAUGUUAUUUUCCACCAUAUUGCCGUCUUUUGGCAAUGUGAGGGCCCGGAAACCUGGCCCUGUCUUCUUGACGAGCAUUCCUAGGGGUCUUUCCCCUCUCGCCAAAGGAAUGCAAGGUCUGUUGAAUGUCGUGAAGGAAGCAGUUCCUCUGGAAGCUUCUUGAAGACAAACAACGUCUGUAGCGACCCUUUGCAGGCAGCGGAACCCCCCACCUGGCGACAGGUGCCUCUGCGGCCAAAAGCCACGUGUAUAAGAUACACCUGCAAAGGCGGCACAACCCCAGUGCCACGUUGUGAGUUGGAUAGUUGUGGAAAGAGUCAAAUGGCUCACCUCAAGCGUAUUCAACAAGGGGCUGAAGGAUGCCCAGAAGGUACCCCAUUGUAUGGGAUCUGAUCUGGGGCCUCGGUGCACAUGCUUUACAUGUGUUUAGUCGAGGUUAAAAAACGUCUAGGCCCCCCGAACCACGGGGACGUGGUUUUCCUUUGAAAAACACGAUGAUAAUAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACA UCUUGCGCAGCCG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGU GGUCUUUGAAUAAAGUCUGAGUGGGCGGCG-CSF Optimized G-CSF cDNA sequence containing a T7 polymerase site, a5007 without a Kozak sequence and Xba1 restriction site: KozakTAATACGACTCACTATA sequence GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAand with ATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGC a humanCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCA alpha-AGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTC globinATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGG 3′UTRGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTCTAGA mRNA sequence (transcribed): 5008GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACA UCUUGCGCAGCCG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGU GGUCUUUGAAUAAAGUCUGAGUGGGCGGCG-CSF Optimized G-CSF cDNA sequence containing a T7 polymerase site,5009 with anIRES, a polyA tail of 80 nucleotides and Asc1 restriction site: IRES, aTAATACGACTCACTATA human GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG alpha-AGCCACC globin TCGTGAGGATCTATTTCCGGTGAATTCCTCGAGACTAGTTCT 3′UTRAGAGCGGCCGCGGATCCCGCCCCTCTCCCTCCCCCCCCCCTA and aACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCG polyATTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCA tail of 80ATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGC nucleotidesATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCACCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAA ACACGATGATAATATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGCGCGCC mRNA sequence (transcribed): 5010GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCUCGUGAGGAUCUAUUUCCGGUGAAUUCCUCGAGACUAGUUCUAGAGCGGCCGCGGAUCCCGCCCCUCUCCCUCCCCCCCCCCUAACGUUACUGGCCGAAGCCGCUUGGAAUAAGGCCGGUGUGCGUUUGUCUAUAUGUUAUUUUCCACCAUAUUGCCGUCUUUUGGCAAUGUGAGGGCCCGGAAACCUGGCCCUGUCUUCUUGACGAGCAUUCCUAGGGGUCUUUCCCCUCUCGCCAAAGGAAUGCAAGGUCUGUUGAAUGUCGUGAAGGAAGCAGUUCCUCUGGAAGCUUCUUGAAGACAAACAACGUCUGUAGCGACCCUUUGCAGGCAGCGGAACCCCCCACCUGGCGACAGGUGCCUCUGCGGCCAAAAGCCACGUGUAUAAGAUACACCUGCAAAGGCGGCACAACCCCAGUGCCACGUUGUGAGUUGGAUAGUUGUGGAAAGAGUCAAAUGGCUCACCUCAAGCGUAUUCAACAAGGGGCUGAAGGAUGCCCAGAAGGUACCCCAUUGUAUGGGAUCUGAUCUGGGGCCUCGGUGCACAUGCUUUACAUGUGUUUAGUCGAGGUUAAAAAACGUCUAGGCCCCCCGAACCACGGGGACGUGGUUUUCCUUUGAAAAACACGAUGAUAAUAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACA UCUUGCGCAGCCG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGU GGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AA G-CSFOptimized G-CSF cDNA sequence containing a T7 polymerase site, 5011without aan IRES sequence, a polyA tail of 80 nucleotides and Asc1 restrictionKozak site: sequence TAATACGACTCACTATA and withGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA an IRES,TCGTGAGGATCTATTTCCGGTGAATTCCTCGAGACTAGTTCT a humanAGAGCGGCCGCGGATCCCGCCCCTCTCCCTCCCCCCCCCCTA alpha-ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCG globinTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCA 3′UTRATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGC and aATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGT polyACTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCT tail of 80TGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCG nucleotidesGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCACCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAA ACACGATGATAATATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGCGCGCC mRNA sequence (transcribed): 5012GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAUCGUGAGGAUCUAUUUCCGGUGAAUUCCUCGAGACUAGUUCUAGAGCGGCCGCGGAUCCCGCCCCUCUCCCUCCCCCCCCCCUAACGUUACUGGCCGAAGCCGCUUGGAAUAAGGCCGGUGUGCGUUUGUCUAUAUGUUAUUUUCCACCAUAUUGCCGUCUUUUGGCAAUGUGAGGGCCCGGAAACCUGGCCCUGUCUUCUUGACGAGCAUUCCUAGGGGUCUUUCCCCUCUCGCCAAAGGAAUGCAAGGUCUGUUGAAUGUCGUGAAGGAAGCAGUUCCUCUGGAAGCUUCUUGAAGACAAACAACGUCUGUAGCGACCCUUUGCAGGCAGCGGAACCCCCCACCUGGCGACAGGUGCCUCUGCGGCCAAAAGCCACGUGUAUAAGAUACACCUGCAAAGGCGGCACAACCCCAGUGCCACGUUGUGAGUUGGAUAGUUGUGGAAAGAGUCAAAUGGCUCACCUCAAGCGUAUUCAACAAGGGGCUGAAGGAUGCCCAGAAGGUACCCCAUUGUAUGGGAUCUGAUCUGGGGCCUCGGUGCACAUGCUUUACAUGUGUUUAGUCGAGGUUAAAAAACGUCUAGGCCCCCCGAACCACGGGGACGUGGUUUUCCUUUGAAAAACACGAUGAUAAUAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACA UCUUGCGCAGCCG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAG-CSF Optimized G-CSF cDNA sequence containing a T7 polymerase site, a5013 with a polyA tail of 80 nucleotides and Asc1 restriction site:human TAATACGACTCACTATA alpha- GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGglobin AGCCACC 3′UTR ATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGC and aCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCA polyAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTC tail of 80ATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGG nucleotidesGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGCGCGCC mRNA sequence (transcribed): 5014GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACA UCUUGCGCAGCCG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAG-CSF Optimized G-CSF cDNA sequence containing a T7 polymerase site, a5015 without a polyA tail of 80 nucleotides and Asc1 restriction site:kozak TAATACGACTCACTATA sequenceGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA and withATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGC a humanCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCA alpha-AGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTC globinATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGG 3′UTRGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATAC and aAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAG polyACTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCA tail of 80GGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGG nucleotidesTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGCGCGCC mRNA sequence (transcribed): 5016GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACA UCUUGCGCAGCCG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

These modified mRNA sequences can include at least one chemicalmodification described herein. The G-CSF modified mRNA sequence can beformulated, using methods described herein and/or known in the art,prior to transfection and/or administration.

The modified mRNA sequence encoding G-CSF can be transfected in vitro tovarious cell types such as HEK293, HeLa, PBMC and BJ fibroblast andthose described in Table 25 using methods disclosed herein and/or areknown in the art. The cells are then analyzed using methods disclosedherein and/or are known in the art to determine the concentration ofG-CSF and/or cell viability.

The modified mRNA sequence encoding G-CSF can also be administered tomammals including mat, rats, non-human primates and humans. The serumand surrounding tissue can be collected at pre-determined intervals andanalyzed using methods disclosed herein and/or are known in the art todetermine the concentration of G-CSF and other pharmacokineticproperties mentioned herein.

Example 32 Modified mRNA Sequences miR-122 Sequences in an Alpha-GlobinUTR

Modified mRNA encoding G-CSF or Factor IX with a mir-122 sequence in ahuman or mouse alpha-globin 3′UTR, and their corresponding cDNAsequences, are shown below in Table 31. In Table 31, the start codon ofeach sequence is underlined.

TABLE 31 G-CSF and FIX Sequences SEQ ID Description Sequence NO:G-CSF with Optimized G-CSF cDNA sequence containing a T7 polymerase site5017 1 miR-122 and Xba1 restriction site: sequence in TAATACGACTCACTATAhuman GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA alpha-globin GAGCCACC3′UTR ATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCTTTGAATAAAGTCTGAGTG GGCGGCTCTAGAmRNA sequence (transcribed): 5018GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGAC AUCUUGCGCAGCCG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCU GAGUGGGCGGC G-CSF withOptimized G-CSF cDNA sequence containing a T7 polymerase site 50191 miR-122 and Xba1 restriction site: seed TAATACGACTCACTATA sequence inGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA human GAGCCACC alpha globinATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGC 3′UTRCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCACACTCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTCTAGA mRNA sequence (transcribed):5020 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGAC AUCUUGCGCAGCCG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACACUCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC G-CSF withOptimized G-CSF cDNA sequence containing a T7 polymerase site 50211 miR-122 and Xba1 restriction site: sequence TAATACGACTCACTATAwithout the GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA seed in GAGCCACChuman ATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGC alpha-globinCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCA 3′UTRAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC TCTAGAmRNA sequence (transcribed): 5022GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGAC AUCUUGCGCAGCCG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCAGUGGUCUUUGAAUAAAGUCUGAGUGG GCGGC G-CSF withOptimized G-CSF cDNA sequence containing a T7 polymerase site 50231 miR-122 and Xba1 restriction site: sequence in TAATACGACTCACTATA mouseGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA alpha-globin GAGCCACC 3′UTRATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTCAAACACCATTGTCACACTCCATGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTC GAGCATGCATCTAGAmRNA sequence (transcribed): 5024GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGAC AUCUUGCGCAGCCG UGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUCAAACACCAUUGUCACACUCCAUGGUCUUUGAAUAAAGCCUGAGUAGGAAG G-CSF withOptimized G-CSF cDNA sequence containing a T7 polymerase site 50251 miR-122 and Xba1 restriction site: seed TAATACGACTCACTATA sequence inGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA mouse GAGCCACC alpha-globinATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGC 3′UTRCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTACACTCCTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA mRNA sequence (transcribed):5026 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGAC AUCUUGCGCAGCCG UGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUACACUCCUGGUCUUUG AAUAAAGCCUGAGUAGGAAG G-CSF withOptimized G-CSF cDNA sequence containing a T7 polymerase site 50271 miR-122 and Xba1 restriction site: sequence TAATACGACTCACTATAwithout the GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA seed in GAGCCACCmouse ATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGC alpha-globinCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCA 3′UTRAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCG TGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTCAAACACCATTGTCATGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATG CATCTAGAmRNA sequence (transcribed): 5028GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGAC AUCUUGCGCAGCCG UGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUCAAACACCAUUGUCAU GGUCUUUGAAUAAAGCCUGAGUAGGAAGFactor IX Optimized Factor IX cDNA sequence containing a T7 polymerase5029 with 1 miR- site and Xba1 restriction site: 122 TAATACGACTCACTATAsequence in GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA human GAGCCACCalpha-globin ATGCAGCGCGTCAACATGATTATGGCCGAATCGCCGGGACT 3′UTRCATCACAATCTGCCTCTTGGGTTATCTCTTGTCGGCAGAATGTACCGTGTTCTTGGATCACGAAAACGCGAACAAAATTCTTAATCGCCCGAAGCGGTATAACTCCGGGAAACTTGAGGAGTTTGTGCAGGGCAATCTTGAACGAGAGTGCATGGAGGAGAAATGCTCCTTTGAGGAGGCGAGGGAAGTGTTTGAAAACACAGAGCGAACAACGGAGTTTTGGAAGCAATACGTAGATGGGGACCAGTGTGAGTCGAATCCGTGCCTCAATGGGGGATCATGTAAAGATGACATCAATAGCTATGAATGCTGGTGCCCGTTTGGGTTTGAAGGGAAGAACTGTGAGCTGGATGTGACGTGCAACATCAAAAACGGACGCTGTGAGCAGTTTTGTAAGAACTCGGCTGACAATAAGGTAGTATGCTCGTGCACAGAGGGATACCGGCTGGCGGAGAACCAAAAATCGTGCGAGCCCGCAGTCCCGTTCCCTTGTGGGAGGGTGAGCGTGTCACAGACTAGCAAGTTGACGAGAGCGGAGACTGTATTCCCCGACGTGGACTACGTCAACAGCACCGAAGCCGAAACAATCCTCGATAACATCACGCAGAGCACTCAGTCCTTCAATGACTTTACGAGGGTCGTAGGTGGTGAGGACGCGAAACCCGGTCAGTTCCCCTGGCAGGTGGTATTGAACGGAAAAGTCGATGCCTTTTGTGGAGGTTCCATTGTCAACGAGAAGTGGATTGTCACAGCGGCACACTGCGTAGAAACAGGAGTGAAAATCACGGTAGTGGCGGGAGAGCATAACATTGAAGAGACAGAGCACACGGAACAAAAGCGAAATGTCATCAGAATCATTCCACACCATAACTATAACGCGGCAATCAATAAGTACAATCACGACATCGCACTTTTGGAGCTTGACGAACCTTTGGTGCTTAATTCGTACGTCACCCCTATTTGTATTGCCGACAAAGAGTATACAAACATCTTCTTGAAATTCGGCTCCGGGTACGTATCGGGCTGGGGCAGAGTGTTCCATAAGGGTAGATCCGCACTGGTGTTGCAATACCTCAGGGTGCCCCTCGTGGATCGAGCCACTTGTCTGCGGTCCACCAAATTCACAATCTACAACAATATGTTCTGTGCGGGATTCCATGAAGGTGGGAGAGATAGCTGCCAGGGAGACTCAGGGGGTCCCCACGTGACGGAAGTCGAGGGGACGTCATTTCTGACGGGAATTATCTCATGGGGAGAGGAATGTGCGATGAAGGGGAAATATGGCATCTACACTAAAGTGTCACGGTATGTCAATTGGATCAAGGAAAAGACGAAACTCACG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCACACTCCAGTGGTCTTTGAATAAAGTCTGAGTG GGCGGCTCTAGAmRNA sequence (transcribed): 5030GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGCAGCGCGUCAACAUGAUUAUGGCCGAAUCGCCGGGACUCAUCACAAUCUGCCUCUUGGGUUAUCUCUUGUCGGCAGAAUGUACCGUGUUCUUGGAUCACGAAAACGCGAACAAAAUUCUUAAUCGCCCGAAGCGGUAUAACUCCGGGAAACUUGAGGAGUUUGUGCAGGGCAAUCUUGAACGAGAGUGCAUGGAGGAGAAAUGCUCCUUUGAGGAGGCGAGGGAAGUGUUUGAAAACACAGAGCGAACAACGGAGUUUUGGAAGCAAUACGUAGAUGGGGACCAGUGUGAGUCGAAUCCGUGCCUCAAUGGGGGAUCAUGUAAAGAUGACAUCAAUAGCUAUGAAUGCUGGUGCCCGUUUGGGUUUGAAGGGAAGAACUGUGAGCUGGAUGUGACGUGCAACAUCAAAAACGGACGCUGUGAGCAGUUUUGUAAGAACUCGGCUGACAAUAAGGUAGUAUGCUCGUGCACAGAGGGAUACCGGCUGGCGGAGAACCAAAAAUCGUGCGAGCCCGCAGUCCCGUUCCCUUGUGGGAGGGUGAGCGUGUCACAGACUAGCAAGUUGACGAGAGCGGAGACUGUAUUCCCCGACGUGGACUACGUCAACAGCACCGAAGCCGAAACAAUCCUCGAUAACAUCACGCAGAGCACUCAGUCCUUCAAUGACUUUACGAGGGUCGUAGGUGGUGAGGACGCGAAACCCGGUCAGUUCCCCUGGCAGGUGGUAUUGAACGGAAAAGUCGAUGCCUUUUGUGGAGGUUCCAUUGUCAACGAGAAGUGGAUUGUCACAGCGGCACACUGCGUAGAAACAGGAGUGAAAAUCACGGUAGUGGCGGGAGAGCAUAACAUUGAAGAGACAGAGCACACGGAACAAAAGCGAAAUGUCAUCAGAAUCAUUCCACACCAUAACUAUAACGCGGCAAUCAAUAAGUACAAUCACGACAUCGCACUUUUGGAGCUUGACGAACCUUUGGUGCUUAAUUCGUACGUCACCCCUAUUUGUAUUGCCGACAAAGAGUAUACAAACAUCUUCUUGAAAUUCGGCUCCGGGUACGUAUCGGGCUGGGGCAGAGUGUUCCAUAAGGGUAGAUCCGCACUGGUGUUGCAAUACCUCAGGGUGCCCCUCGUGGAUCGAGCCACUUGUCUGCGGUCCACCAAAUUCACAAUCUACAACAAUAUGUUCUGUGCGGGAUUCCAUGAAGGUGGGAGAGAUAGCUGCCAGGGAGACUCAGGGGGUCCCCACGUGACGGAAGUCGAGGGGACGUCAUUUCUGACGGGAAUUAUCUCAUGGGGAGAGGAAUGUGCGAUGAAGGGGAAAUAUGGCAUCUACACUAAAGUGUCACGGUAUGUCAAUUGGAUCAAGGAAAAGACGAAACUCACG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCACACUCCAGUGGUCUUUGAAUAAAGUCU GAGUGGGCGGC Factor IXOptimized Factor IX cDNA sequence containing a T7 polymerase site 5031with 1 miR- and Xba1 restriction site: 122 seed TAATACGACTCACTATAsequence in GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA human GAGCCACCalpha-globin ATGCAGCGCGTCAACATGATTATGGCCGAATCGCCGGGACT 3′UTRCATCACAATCTGCCTCTTGGGTTATCTCTTGTCGGCAGAATGTACCGTGTTCTTGGATCACGAAAACGCGAACAAAATTCTTAATCGCCCGAAGCGGTATAACTCCGGGAAACTTGAGGAGTTTGTGCAGGGCAATCTTGAACGAGAGTGCATGGAGGAGAAATGCTCCTTTGAGGAGGCGAGGGAAGTGTTTGAAAACACAGAGCGAACAACGGAGTTTTGGAAGCAATACGTAGATGGGGACCAGTGTGAGTCGAATCCGTGCCTCAATGGGGGATCATGTAAAGATGACATCAATAGCTATGAATGCTGGTGCCCGTTTGGGTTTGAAGGGAAGAACTGTGAGCTGGATGTGACGTGCAACATCAAAAACGGACGCTGTGAGCAGTTTTGTAAGAACTCGGCTGACAATAAGGTAGTATGCTCGTGCACAGAGGGATACCGGCTGGCGGAGAACCAAAAATCGTGCGAGCCCGCAGTCCCGTTCCCTTGTGGGAGGGTGAGCGTGTCACAGACTAGCAAGTTGACGAGAGCGGAGACTGTATTCCCCGACGTGGACTACGTCAACAGCACCGAAGCCGAAACAATCCTCGATAACATCACGCAGAGCACTCAGTCCTTCAATGACTTTACGAGGGTCGTAGGTGGTGAGGACGCGAAACCCGGTCAGTTCCCCTGGCAGGTGGTATTGAACGGAAAAGTCGATGCCTTTTGTGGAGGTTCCATTGTCAACGAGAAGTGGATTGTCACAGCGGCACACTGCGTAGAAACAGGAGTGAAAATCACGGTAGTGGCGGGAGAGCATAACATTGAAGAGACAGAGCACACGGAACAAAAGCGAAATGTCATCAGAATCATTCCACACCATAACTATAACGCGGCAATCAATAAGTACAATCACGACATCGCACTTTTGGAGCTTGACGAACCTTTGGTGCTTAATTCGTACGTCACCCCTATTTGTATTGCCGACAAAGAGTATACAAACATCTTCTTGAAATTCGGCTCCGGGTACGTATCGGGCTGGGGCAGAGTGTTCCATAAGGGTAGATCCGCACTGGTGTTGCAATACCTCAGGGTGCCCCTCGTGGATCGAGCCACTTGTCTGCGGTCCACCAAATTCACAATCTACAACAATATGTTCTGTGCGGGATTCCATGAAGGTGGGAGAGATAGCTGCCAGGGAGACTCAGGGGGTCCCCACGTGACGGAAGTCGAGGGGACGTCATTTCTGACGGGAATTATCTCATGGGGAGAGGAATGTGCGATGAAGGGGAAATATGGCATCTACACTAAAGTGTCACGGTATGTCAATTGGATCAAGGAAAAGACGAAACTCACG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCACACTCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTCTAGA mRNA sequence (transcribed):5032 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGCAGCGCGUCAACAUGAUUAUGGCCGAAUCGCCGGGACUCAUCACAAUCUGCCUCUUGGGUUAUCUCUUGUCGGCAGAAUGUACCGUGUUCUUGGAUCACGAAAACGCGAACAAAAUUCUUAAUCGCCCGAAGCGGUAUAACUCCGGGAAACUUGAGGAGUUUGUGCAGGGCAAUCUUGAACGAGAGUGCAUGGAGGAGAAAUGCUCCUUUGAGGAGGCGAGGGAAGUGUUUGAAAACACAGAGCGAACAACGGAGUUUUGGAAGCAAUACGUAGAUGGGGACCAGUGUGAGUCGAAUCCGUGCCUCAAUGGGGGAUCAUGUAAAGAUGACAUCAAUAGCUAUGAAUGCUGGUGCCCGUUUGGGUUUGAAGGGAAGAACUGUGAGCUGGAUGUGACGUGCAACAUCAAAAACGGACGCUGUGAGCAGUUUUGUAAGAACUCGGCUGACAAUAAGGUAGUAUGCUCGUGCACAGAGGGAUACCGGCUGGCGGAGAACCAAAAAUCGUGCGAGCCCGCAGUCCCGUUCCCUUGUGGGAGGGUGAGCGUGUCACAGACUAGCAAGUUGACGAGAGCGGAGACUGUAUUCCCCGACGUGGACUACGUCAACAGCACCGAAGCCGAAACAAUCCUCGAUAACAUCACGCAGAGCACUCAGUCCUUCAAUGACUUUACGAGGGUCGUAGGUGGUGAGGACGCGAAACCCGGUCAGUUCCCCUGGCAGGUGGUAUUGAACGGAAAAGUCGAUGCCUUUUGUGGAGGUUCCAUUGUCAACGAGAAGUGGAUUGUCACAGCGGCACACUGCGUAGAAACAGGAGUGAAAAUCACGGUAGUGGCGGGAGAGCAUAACAUUGAAGAGACAGAGCACACGGAACAAAAGCGAAAUGUCAUCAGAAUCAUUCCACACCAUAACUAUAACGCGGCAAUCAAUAAGUACAAUCACGACAUCGCACUUUUGGAGCUUGACGAACCUUUGGUGCUUAAUUCGUACGUCACCCCUAUUUGUAUUGCCGACAAAGAGUAUACAAACAUCUUCUUGAAAUUCGGCUCCGGGUACGUAUCGGGCUGGGGCAGAGUGUUCCAUAAGGGUAGAUCCGCACUGGUGUUGCAAUACCUCAGGGUGCCCCUCGUGGAUCGAGCCACUUGUCUGCGGUCCACCAAAUUCACAAUCUACAACAAUAUGUUCUGUGCGGGAUUCCAUGAAGGUGGGAGAGAUAGCUGCCAGGGAGACUCAGGGGGUCCCCACGUGACGGAAGUCGAGGGGACGUCAUUUCUGACGGGAAUUAUCUCAUGGGGAGAGGAAUGUGCGAUGAAGGGGAAAUAUGGCAUCUACACUAAAGUGUCACGGUAUGUCAAUUGGAUCAAGGAAAAGACGAAACUCACG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCACACUCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC Factor IXOptimized Factor IX cDNA sequence containing a T7 polymerase site 5033with 1 miR- and Xba1 restriction site: 122 TAATACGACTCACTATA sequenceGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA without the GAGCCACC seed inATGCAGCGCGTCAACATGATTATGGCCGAATCGCCGGGACT humanCATCACAATCTGCCTCTTGGGTTATCTCTTGTCGGCAGAATG alpha-globinTACCGTGTTCTTGGATCACGAAAACGCGAACAAAATTCTTA 3′UTRATCGCCCGAAGCGGTATAACTCCGGGAAACTTGAGGAGTTTGTGCAGGGCAATCTTGAACGAGAGTGCATGGAGGAGAAATGCTCCTTTGAGGAGGCGAGGGAAGTGTTTGAAAACACAGAGCGAACAACGGAGTTTTGGAAGCAATACGTAGATGGGGACCAGTGTGAGTCGAATCCGTGCCTCAATGGGGGATCATGTAAAGATGACATCAATAGCTATGAATGCTGGTGCCCGTTTGGGTTTGAAGGGAAGAACTGTGAGCTGGATGTGACGTGCAACATCAAAAACGGACGCTGTGAGCAGTTTTGTAAGAACTCGGCTGACAATAAGGTAGTATGCTCGTGCACAGAGGGATACCGGCTGGCGGAGAACCAAAAATCGTGCGAGCCCGCAGTCCCGTTCCCTTGTGGGAGGGTGAGCGTGTCACAGACTAGCAAGTTGACGAGAGCGGAGACTGTATTCCCCGACGTGGACTACGTCAACAGCACCGAAGCCGAAACAATCCTCGATAACATCACGCAGAGCACTCAGTCCTTCAATGACTTTACGAGGGTCGTAGGTGGTGAGGACGCGAAACCCGGTCAGTTCCCCTGGCAGGTGGTATTGAACGGAAAAGTCGATGCCTTTTGTGGAGGTTCCATTGTCAACGAGAAGTGGATTGTCACAGCGGCACACTGCGTAGAAACAGGAGTGAAAATCACGGTAGTGGCGGGAGAGCATAACATTGAAGAGACAGAGCACACGGAACAAAAGCGAAATGTCATCAGAATCATTCCACACCATAACTATAACGCGGCAATCAATAAGTACAATCACGACATCGCACTTTTGGAGCTTGACGAACCTTTGGTGCTTAATTCGTACGTCACCCCTATTTGTATTGCCGACAAAGAGTATACAAACATCTTCTTGAAATTCGGCTCCGGGTACGTATCGGGCTGGGGCAGAGTGTTCCATAAGGGTAGATCCGCACTGGTGTTGCAATACCTCAGGGTGCCCCTCGTGGATCGAGCCACTTGTCTGCGGTCCACCAAATTCACAATCTACAACAATATGTTCTGTGCGGGATTCCATGAAGGTGGGAGAGATAGCTGCCAGGGAGACTCAGGGGGTCCCCACGTGACGGAAGTCGAGGGGACGTCATTTCTGACGGGAATTATCTCATGGGGAGAGGAATGTGCGATGAAGGGGAAATATGGCATCTACACTAAAGTGTCACGGTATGTCAATTGGATCAAGGAAAAGACGAAACTCACG TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCCAAACACCATTGTCAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC TCTAGAmRNA sequence (transcribed): 5034GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGCAGCGCGUCAACAUGAUUAUGGCCGAAUCGCCGGGACUCAUCACAAUCUGCCUCUUGGGUUAUCUCUUGUCGGCAGAAUGUACCGUGUUCUUGGAUCACGAAAACGCGAACAAAAUUCUUAAUCGCCCGAAGCGGUAUAACUCCGGGAAACUUGAGGAGUUUGUGCAGGGCAAUCUUGAACGAGAGUGCAUGGAGGAGAAAUGCUCCUUUGAGGAGGCGAGGGAAGUGUUUGAAAACACAGAGCGAACAACGGAGUUUUGGAAGCAAUACGUAGAUGGGGACCAGUGUGAGUCGAAUCCGUGCCUCAAUGGGGGAUCAUGUAAAGAUGACAUCAAUAGCUAUGAAUGCUGGUGCCCGUUUGGGUUUGAAGGGAAGAACUGUGAGCUGGAUGUGACGUGCAACAUCAAAAACGGACGCUGUGAGCAGUUUUGUAAGAACUCGGCUGACAAUAAGGUAGUAUGCUCGUGCACAGAGGGAUACCGGCUGGCGGAGAACCAAAAAUCGUGCGAGCCCGCAGUCCCGUUCCCUUGUGGGAGGGUGAGCGUGUCACAGACUAGCAAGUUGACGAGAGCGGAGACUGUAUUCCCCGACGUGGACUACGUCAACAGCACCGAAGCCGAAACAAUCCUCGAUAACAUCACGCAGAGCACUCAGUCCUUCAAUGACUUUACGAGGGUCGUAGGUGGUGAGGACGCGAAACCCGGUCAGUUCCCCUGGCAGGUGGUAUUGAACGGAAAAGUCGAUGCCUUUUGUGGAGGUUCCAUUGUCAACGAGAAGUGGAUUGUCACAGCGGCACACUGCGUAGAAACAGGAGUGAAAAUCACGGUAGUGGCGGGAGAGCAUAACAUUGAAGAGACAGAGCACACGGAACAAAAGCGAAAUGUCAUCAGAAUCAUUCCACACCAUAACUAUAACGCGGCAAUCAAUAAGUACAAUCACGACAUCGCACUUUUGGAGCUUGACGAACCUUUGGUGCUUAAUUCGUACGUCACCCCUAUUUGUAUUGCCGACAAAGAGUAUACAAACAUCUUCUUGAAAUUCGGCUCCGGGUACGUAUCGGGCUGGGGCAGAGUGUUCCAUAAGGGUAGAUCCGCACUGGUGUUGCAAUACCUCAGGGUGCCCCUCGUGGAUCGAGCCACUUGUCUGCGGUCCACCAAAUUCACAAUCUACAACAAUAUGUUCUGUGCGGGAUUCCAUGAAGGUGGGAGAGAUAGCUGCCAGGGAGACUCAGGGGGUCCCCACGUGACGGAAGUCGAGGGGACGUCAUUUCUGACGGGAAUUAUCUCAUGGGGAGAGGAAUGUGCGAUGAAGGGGAAAUAUGGCAUCUACACUAAAGUGUCACGGUAUGUCAAUUGGAUCAAGGAAAAGACGAAACUCACG UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCAAACACCAUUGUCAGUGGUCUUUGAAUAAAGUCUGAGUGG GCGGC Factor IXOptimized Factor IX cDNA sequence containing a T7 polymerase site 5035with 1 miR- and Xba1 restriction site: 122 TAATACGACTCACTATA sequence inGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA mouse GAGCCACC alpha-globinATGCAGCGCGTCAACATGATTATGGCCGAATCGCCGGGACT 3′UTRCATCACAATCTGCCTCTTGGGTTATCTCTTGTCGGCAGAATGTACCGTGTTCTTGGATCACGAAAACGCGAACAAAATTCTTAATCGCCCGAAGCGGTATAACTCCGGGAAACTTGAGGAGTTTGTGCAGGGCAATCTTGAACGAGAGTGCATGGAGGAGAAATGCTCCTTTGAGGAGGCGAGGGAAGTGTTTGAAAACACAGAGCGAACAACGGAGTTTTGGAAGCAATACGTAGATGGGGACCAGTGTGAGTCGAATCCGTGCCTCAATGGGGGATCATGTAAAGATGACATCAATAGCTATGAATGCTGGTGCCCGTTTGGGTTTGAAGGGAAGAACTGTGAGCTGGATGTGACGTGCAACATCAAAAACGGACGCTGTGAGCAGTTTTGTAAGAACTCGGCTGACAATAAGGTAGTATGCTCGTGCACAGAGGGATACCGGCTGGCGGAGAACCAAAAATCGTGCGAGCCCGCAGTCCCGTTCCCTTGTGGGAGGGTGAGCGTGTCACAGACTAGCAAGTTGACGAGAGCGGAGACTGTATTCCCCGACGTGGACTACGTCAACAGCACCGAAGCCGAAACAATCCTCGATAACATCACGCAGAGCACTCAGTCCTTCAATGACTTTACGAGGGTCGTAGGTGGTGAGGACGCGAAACCCGGTCAGTTCCCCTGGCAGGTGGTATTGAACGGAAAAGTCGATGCCTTTTGTGGAGGTTCCATTGTCAACGAGAAGTGGATTGTCACAGCGGCACACTGCGTAGAAACAGGAGTGAAAATCACGGTAGTGGCGGGAGAGCATAACATTGAAGAGACAGAGCACACGGAACAAAAGCGAAATGTCATCAGAATCATTCCACACCATAACTATAACGCGGCAATCAATAAGTACAATCACGACATCGCACTTTTGGAGCTTGACGAACCTTTGGTGCTTAATTCGTACGTCACCCCTATTTGTATTGCCGACAAAGAGTATACAAACATCTTCTTGAAATTCGGCTCCGGGTACGTATCGGGCTGGGGCAGAGTGTTCCATAAGGGTAGATCCGCACTGGTGTTGCAATACCTCAGGGTGCCCCTCGTGGATCGAGCCACTTGTCTGCGGTCCACCAAATTCACAATCTACAACAATATGTTCTGTGCGGGATTCCATGAAGGTGGGAGAGATAGCTGCCAGGGAGACTCAGGGGGTCCCCACGTGACGGAAGTCGAGGGGACGTCATTTCTGACGGGAATTATCTCATGGGGAGAGGAATGTGCGATGAAGGGGAAATATGGCATCTACACTAAAGTGTCACGGTATGTCAATTGGATCAAGGAAAAGACGAAACTCACG TGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTCAAACACCATTGTCACACTCCATGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTC GAGCATGCATCTAGAmRNA sequence (transcribed): 5036GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGCAGCGCGUCAACAUGAUUAUGGCCGAAUCGCCGGGACUCAUCACAAUCUGCCUCUUGGGUUAUCUCUUGUCGGCAGAAUGUACCGUGUUCUUGGAUCACGAAAACGCGAACAAAAUUCUUAAUCGCCCGAAGCGGUAUAACUCCGGGAAACUUGAGGAGUUUGUGCAGGGCAAUCUUGAACGAGAGUGCAUGGAGGAGAAAUGCUCCUUUGAGGAGGCGAGGGAAGUGUUUGAAAACACAGAGCGAACAACGGAGUUUUGGAAGCAAUACGUAGAUGGGGACCAGUGUGAGUCGAAUCCGUGCCUCAAUGGGGGAUCAUGUAAAGAUGACAUCAAUAGCUAUGAAUGCUGGUGCCCGUUUGGGUUUGAAGGGAAGAACUGUGAGCUGGAUGUGACGUGCAACAUCAAAAACGGACGCUGUGAGCAGUUUUGUAAGAACUCGGCUGACAAUAAGGUAGUAUGCUCGUGCACAGAGGGAUACCGGCUGGCGGAGAACCAAAAAUCGUGCGAGCCCGCAGUCCCGUUCCCUUGUGGGAGGGUGAGCGUGUCACAGACUAGCAAGUUGACGAGAGCGGAGACUGUAUUCCCCGACGUGGACUACGUCAACAGCACCGAAGCCGAAACAAUCCUCGAUAACAUCACGCAGAGCACUCAGUCCUUCAAUGACUUUACGAGGGUCGUAGGUGGUGAGGACGCGAAACCCGGUCAGUUCCCCUGGCAGGUGGUAUUGAACGGAAAAGUCGAUGCCUUUUGUGGAGGUUCCAUUGUCAACGAGAAGUGGAUUGUCACAGCGGCACACUGCGUAGAAACAGGAGUGAAAAUCACGGUAGUGGCGGGAGAGCAUAACAUUGAAGAGACAGAGCACACGGAACAAAAGCGAAAUGUCAUCAGAAUCAUUCCACACCAUAACUAUAACGCGGCAAUCAAUAAGUACAAUCACGACAUCGCACUUUUGGAGCUUGACGAACCUUUGGUGCUUAAUUCGUACGUCACCCCUAUUUGUAUUGCCGACAAAGAGUAUACAAACAUCUUCUUGAAAUUCGGCUCCGGGUACGUAUCGGGCUGGGGCAGAGUGUUCCAUAAGGGUAGAUCCGCACUGGUGUUGCAAUACCUCAGGGUGCCCCUCGUGGAUCGAGCCACUUGUCUGCGGUCCACCAAAUUCACAAUCUACAACAAUAUGUUCUGUGCGGGAUUCCAUGAAGGUGGGAGAGAUAGCUGCCAGGGAGACUCAGGGGGUCCCCACGUGACGGAAGUCGAGGGGACGUCAUUUCUGACGGGAAUUAUCUCAUGGGGAGAGGAAUGUGCGAUGAAGGGGAAAUAUGGCAUCUACACUAAAGUGUCACGGUAUGUCAAUUGGAUCAAGGAAAAGACGAAACUCACG UGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUCAAACACCAUUGUCACACUCCAUGGUCUUUGAAUAAAGCCUGAGUAGGAAG Factor IXOptimized Factor IX cDNA sequence containing a T7 polymerase site 5037with 1 miR- and Xba1 restriction site: 122 seed TAATACGACTCACTATAsequence in GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA mouse GAGCCACCalpha-globin ATGCAGCGCGTCAACATGATTATGGCCGAATCGCCGGGACT 3′UTRCATCACAATCTGCCTCTTGGGTTATCTCTTGTCGGCAGAATGTACCGTGTTCTTGGATCACGAAAACGCGAACAAAATTCTTAATCGCCCGAAGCGGTATAACTCCGGGAAACTTGAGGAGTTTGTGCAGGGCAATCTTGAACGAGAGTGCATGGAGGAGAAATGCTCCTTTGAGGAGGCGAGGGAAGTGTTTGAAAACACAGAGCGAACAACGGAGTTTTGGAAGCAATACGTAGATGGGGACCAGTGTGAGTCGAATCCGTGCCTCAATGGGGGATCATGTAAAGATGACATCAATAGCTATGAATGCTGGTGCCCGTTTGGGTTTGAAGGGAAGAACTGTGAGCTGGATGTGACGTGCAACATCAAAAACGGACGCTGTGAGCAGTTTTGTAAGAACTCGGCTGACAATAAGGTAGTATGCTCGTGCACAGAGGGATACCGGCTGGCGGAGAACCAAAAATCGTGCGAGCCCGCAGTCCCGTTCCCTTGTGGGAGGGTGAGCGTGTCACAGACTAGCAAGTTGACGAGAGCGGAGACTGTATTCCCCGACGTGGACTACGTCAACAGCACCGAAGCCGAAACAATCCTCGATAACATCACGCAGAGCACTCAGTCCTTCAATGACTTTACGAGGGTCGTAGGTGGTGAGGACGCGAAACCCGGTCAGTTCCCCTGGCAGGTGGTATTGAACGGAAAAGTCGATGCCTTTTGTGGAGGTTCCATTGTCAACGAGAAGTGGATTGTCACAGCGGCACACTGCGTAGAAACAGGAGTGAAAATCACGGTAGTGGCGGGAGAGCATAACATTGAAGAGACAGAGCACACGGAACAAAAGCGAAATGTCATCAGAATCATTCCACACCATAACTATAACGCGGCAATCAATAAGTACAATCACGACATCGCACTTTTGGAGCTTGACGAACCTTTGGTGCTTAATTCGTACGTCACCCCTATTTGTATTGCCGACAAAGAGTATACAAACATCTTCTTGAAATTCGGCTCCGGGTACGTATCGGGCTGGGGCAGAGTGTTCCATAAGGGTAGATCCGCACTGGTGTTGCAATACCTCAGGGTGCCCCTCGTGGATCGAGCCACTTGTCTGCGGTCCACCAAATTCACAATCTACAACAATATGTTCTGTGCGGGATTCCATGAAGGTGGGAGAGATAGCTGCCAGGGAGACTCAGGGGGTCCCCACGTGACGGAAGTCGAGGGGACGTCATTTCTGACGGGAATTATCTCATGGGGAGAGGAATGTGCGATGAAGGGGAAATATGGCATCTACACTAAAGTGTCACGGTATGTCAATTGGATCAAGGAAAAGACGAAACTCACG TGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTACACTCCTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA mRNA sequence (transcribed):5038 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGCAGCGCGUCAACAUGAUUAUGGCCGAAUCGCCGGGACUCAUCACAAUCUGCCUCUUGGGUUAUCUCUUGUCGGCAGAAUGUACCGUGUUCUUGGAUCACGAAAACGCGAACAAAAUUCUUAAUCGCCCGAAGCGGUAUAACUCCGGGAAACUUGAGGAGUUUGUGCAGGGCAAUCUUGAACGAGAGUGCAUGGAGGAGAAAUGCUCCUUUGAGGAGGCGAGGGAAGUGUUUGAAAACACAGAGCGAACAACGGAGUUUUGGAAGCAAUACGUAGAUGGGGACCAGUGUGAGUCGAAUCCGUGCCUCAAUGGGGGAUCAUGUAAAGAUGACAUCAAUAGCUAUGAAUGCUGGUGCCCGUUUGGGUUUGAAGGGAAGAACUGUGAGCUGGAUGUGACGUGCAACAUCAAAAACGGACGCUGUGAGCAGUUUUGUAAGAACUCGGCUGACAAUAAGGUAGUAUGCUCGUGCACAGAGGGAUACCGGCUGGCGGAGAACCAAAAAUCGUGCGAGCCCGCAGUCCCGUUCCCUUGUGGGAGGGUGAGCGUGUCACAGACUAGCAAGUUGACGAGAGCGGAGACUGUAUUCCCCGACGUGGACUACGUCAACAGCACCGAAGCCGAAACAAUCCUCGAUAACAUCACGCAGAGCACUCAGUCCUUCAAUGACUUUACGAGGGUCGUAGGUGGUGAGGACGCGAAACCCGGUCAGUUCCCCUGGCAGGUGGUAUUGAACGGAAAAGUCGAUGCCUUUUGUGGAGGUUCCAUUGUCAACGAGAAGUGGAUUGUCACAGCGGCACACUGCGUAGAAACAGGAGUGAAAAUCACGGUAGUGGCGGGAGAGCAUAACAUUGAAGAGACAGAGCACACGGAACAAAAGCGAAAUGUCAUCAGAAUCAUUCCACACCAUAACUAUAACGCGGCAAUCAAUAAGUACAAUCACGACAUCGCACUUUUGGAGCUUGACGAACCUUUGGUGCUUAAUUCGUACGUCACCCCUAUUUGUAUUGCCGACAAAGAGUAUACAAACAUCUUCUUGAAAUUCGGCUCCGGGUACGUAUCGGGCUGGGGCAGAGUGUUCCAUAAGGGUAGAUCCGCACUGGUGUUGCAAUACCUCAGGGUGCCCCUCGUGGAUCGAGCCACUUGUCUGCGGUCCACCAAAUUCACAAUCUACAACAAUAUGUUCUGUGCGGGAUUCCAUGAAGGUGGGAGAGAUAGCUGCCAGGGAGACUCAGGGGGUCCCCACGUGACGGAAGUCGAGGGGACGUCAUUUCUGACGGGAAUUAUCUCAUGGGGAGAGGAAUGUGCGAUGAAGGGGAAAUAUGGCAUCUACACUAAAGUGUCACGGUAUGUCAAUUGGAUCAAGGAAAAGACGAAACUCACG UGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUACACUCCUGGUCUUUG AAUAAAGCCUGAGUAGGAAG Factor IXOptimized Factor IX cDNA sequence containing a T7 polymerase site 5039with 1 miR- and Xba1 restriction site: 122 TAATACGACTCACTATA sequenceGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAA without the GAGCCACC seed inATGCAGCGCGTCAACATGATTATGGCCGAATCGCCGGGACT mouseCATCACAATCTGCCTCTTGGGTTATCTCTTGTCGGCAGAATG alpha-globinTACCGTGTTCTTGGATCACGAAAACGCGAACAAAATTCTTA 3′UTRATCGCCCGAAGCGGTATAACTCCGGGAAACTTGAGGAGTTTGTGCAGGGCAATCTTGAACGAGAGTGCATGGAGGAGAAATGCTCCTTTGAGGAGGCGAGGGAAGTGTTTGAAAACACAGAGCGAACAACGGAGTTTTGGAAGCAATACGTAGATGGGGACCAGTGTGAGTCGAATCCGTGCCTCAATGGGGGATCATGTAAAGATGACATCAATAGCTATGAATGCTGGTGCCCGTTTGGGTTTGAAGGGAAGAACTGTGAGCTGGATGTGACGTGCAACATCAAAAACGGACGCTGTGAGCAGTTTTGTAAGAACTCGGCTGACAATAAGGTAGTATGCTCGTGCACAGAGGGATACCGGCTGGCGGAGAACCAAAAATCGTGCGAGCCCGCAGTCCCGTTCCCTTGTGGGAGGGTGAGCGTGTCACAGACTAGCAAGTTGACGAGAGCGGAGACTGTATTCCCCGACGTGGACTACGTCAACAGCACCGAAGCCGAAACAATCCTCGATAACATCACGCAGAGCACTCAGTCCTTCAATGACTTTACGAGGGTCGTAGGTGGTGAGGACGCGAAACCCGGTCAGTTCCCCTGGCAGGTGGTATTGAACGGAAAAGTCGATGCCTTTTGTGGAGGTTCCATTGTCAACGAGAAGTGGATTGTCACAGCGGCACACTGCGTAGAAACAGGAGTGAAAATCACGGTAGTGGCGGGAGAGCATAACATTGAAGAGACAGAGCACACGGAACAAAAGCGAAATGTCATCAGAATCATTCCACACCATAACTATAACGCGGCAATCAATAAGTACAATCACGACATCGCACTTTTGGAGCTTGACGAACCTTTGGTGCTTAATTCGTACGTCACCCCTATTTGTATTGCCGACAAAGAGTATACAAACATCTTCTTGAAATTCGGCTCCGGGTACGTATCGGGCTGGGGCAGAGTGTTCCATAAGGGTAGATCCGCACTGGTGTTGCAATACCTCAGGGTGCCCCTCGTGGATCGAGCCACTTGTCTGCGGTCCACCAAATTCACAATCTACAACAATATGTTCTGTGCGGGATTCCATGAAGGTGGGAGAGATAGCTGCCAGGGAGACTCAGGGGGTCCCCACGTGACGGAAGTCGAGGGGACGTCATTTCTGACGGGAATTATCTCATGGGGAGAGGAATGTGCGATGAAGGGGAAATATGGCATCTACACTAAAGTGTCACGGTATGTCAATTGGATCAAGGAAAAGACGAAACTCACG TGATAATAGGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTC TCTCCCTTGCACCTGTACCTCTCAAACACCATTGTCATGGTCTTTGAATAAAGCCTGAGTAGG AAGGCGGCCGCTCGAGCATGCATCTAGAmRNA sequence (transcribed): 5040GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA GAGCCACCAUGCAGCGCGUCAACAUGAUUAUGGCCGAAUCGCCGGGACUCAUCACAAUCUGCCUCUUGGGUUAUCUCUUGUCGGCAGAAUGUACCGUGUUCUUGGAUCACGAAAACGCGAACAAAAUUCUUAAUCGCCCGAAGCGGUAUAACUCCGGGAAACUUGAGGAGUUUGUGCAGGGCAAUCUUGAACGAGAGUGCAUGGAGGAGAAAUGCUCCUUUGAGGAGGCGAGGGAAGUGUUUGAAAACACAGAGCGAACAACGGAGUUUUGGAAGCAAUACGUAGAUGGGGACCAGUGUGAGUCGAAUCCGUGCCUCAAUGGGGGAUCAUGUAAAGAUGACAUCAAUAGCUAUGAAUGCUGGUGCCCGUUUGGGUUUGAAGGGAAGAACUGUGAGCUGGAUGUGACGUGCAACAUCAAAAACGGACGCUGUGAGCAGUUUUGUAAGAACUCGGCUGACAAUAAGGUAGUAUGCUCGUGCACAGAGGGAUACCGGCUGGCGGAGAACCAAAAAUCGUGCGAGCCCGCAGUCCCGUUCCCUUGUGGGAGGGUGAGCGUGUCACAGACUAGCAAGUUGACGAGAGCGGAGACUGUAUUCCCCGACGUGGACUACGUCAACAGCACCGAAGCCGAAACAAUCCUCGAUAACAUCACGCAGAGCACUCAGUCCUUCAAUGACUUUACGAGGGUCGUAGGUGGUGAGGACGCGAAACCCGGUCAGUUCCCCUGGCAGGUGGUAUUGAACGGAAAAGUCGAUGCCUUUUGUGGAGGUUCCAUUGUCAACGAGAAGUGGAUUGUCACAGCGGCACACUGCGUAGAAACAGGAGUGAAAAUCACGGUAGUGGCGGGAGAGCAUAACAUUGAAGAGACAGAGCACACGGAACAAAAGCGAAAUGUCAUCAGAAUCAUUCCACACCAUAACUAUAACGCGGCAAUCAAUAAGUACAAUCACGACAUCGCACUUUUGGAGCUUGACGAACCUUUGGUGCUUAAUUCGUACGUCACCCCUAUUUGUAUUGCCGACAAAGAGUAUACAAACAUCUUCUUGAAAUUCGGCUCCGGGUACGUAUCGGGCUGGGGCAGAGUGUUCCAUAAGGGUAGAUCCGCACUGGUGUUGCAAUACCUCAGGGUGCCCCUCGUGGAUCGAGCCACUUGUCUGCGGUCCACCAAAUUCACAAUCUACAACAAUAUGUUCUGUGCGGGAUUCCAUGAAGGUGGGAGAGAUAGCUGCCAGGGAGACUCAGGGGGUCCCCACGUGACGGAAGUCGAGGGGACGUCAUUUCUGACGGGAAUUAUCUCAUGGGGAGAGGAAUGUGCGAUGAAGGGGAAAUAUGGCAUCUACACUAAAGUGUCACGGUAUGUCAAUUGGAUCAAGGAAAAGACGAAACUCACG UGAUAAUAGGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCU UCUCUCCCUUGCACCUGUACCUCUCAAACACCAUUGUCAUGGUCUUUGAAUAAAGCCUGAGUA GGAAG

These modified mRNA sequences can include at least one chemicalmodification described herein. The G-CSF and/or Factor IX modified mRNAsequence can be formulated, using methods described herein and/or knownin the art, prior to transfection and/or administration.

The modified mRNA sequence encoding G-CSF or Factor IX can betransfected in vitro to various cell types such as HEK293, HeLa, PBMCand BJ fibroblast and those described in Table 25 using methodsdisclosed herein and/or are known in the art. The cells are thenanalyzed using methods disclosed herein and/or are known in the art todetermine the concentration of G-CSF or Factor IX and/or cell viability.

The modified mRNA sequence encoding G-CSF or Factor IX can also beadministered to mammals including mat, rats, non-human primates andhumans. The serum, surrounding tissue and organs can be collected atpre-determined intervals and analyzed using methods disclosed hereinand/or are known in the art to determine the concentration of G-CSF orFactor IX and other pharmacokinetic properties mentioned herein.

Example 33 Microphysiological Systems

The polynucleotides, primary constructs and/or mmRNA described hereinare formulated using one of the methods described herein such as inbuffer, lipid nanoparticles and PLGA. These formulations are thenadministered to or contacted with microphysiological systems createdfrom organ chips as described in International Publication Nos.WO2013086502, WO2013086486 and WO2013086505, the contents of each ofwhich are herein incorporated by reference in its entirety.

Example 34 Translation Enhancing Elements (TEEs) in Untranslated Regions

The 5′ and/or 3′ untranslated regions (UTRs) in the polynucleotides,primary constructs and/or mmRNA described herein may include at leastone translation enhancing element (TEE). Such TEE which may be includedin the 5′UTR and/or 3′UTR include, but are not limited to, those listedin Table 32, including portion and/or fragments thereof. The TEEsequence may include at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 99% or more than 99% of the TEE sequences disclosed inTable 28 and/or the TEE sequence may include a 5-30 nucleotide fragment,a 5-25 nucleotide fragment, a 5-20 nucleotide fragment, a 5-15nucleotide fragment, a 5-10 nucleotide fragment of the TEE sequencesdisclosed in Table 32.

TABLE 32 TEE Sequences TEE SEQ ID Identifier Sequence NO TEE-001MSCSGCNGMWA 5041 TEE-002 RNSGAGMGRMR 5042 TEE-003 RNSGAGMGRMRRR 5043TEE-004 RMSCSGCNGMWR 5044 TEE-005 GCGAGAGAA — TEE-006 GGGAGCGAA —TEE-007 GCGAGAGGA — TEE-008 GCGAGCGGA — TEE-009 CGGAGCGAA — TEE-010CGGAGCGGA — TEE-011 ACGAGAGGA — TEE-012 ACGAGCGGA — TEE-013 GACGAGAGGA5045 TEE-014 GACGAGAGAA 5046 TEE-015 AGCGAGCG — TEE-016 AGGAGAGGA —TEE-017 GCCGAGAGA — TEE-018 CGAGAGGCA — TEE-019 GAGAGGAGC — TEE-020CGCGGCGGA — TEE-021 CGCCGCCGC — TEE-022 GCGGCTGAA — TEE-023 CCGGCTGAA —TEE-024 CGCCGCTGAA 5047 TEE-025 CGCCGCGGAA 5048 TEE-026 CGCCGCCGAA 5049TEE-027 CCCGCGGAA — TEE-028 CCCGCCGAA — TEE-029 CCCGCTGAA — TEE-030CCCGGCGGA — TEE-031 CGCGGCTGA — TEE-032 CGGCTGCTA — TEE-033 CCCGGCGGA —TEE-034 AGCCGCCGCA 5050 TEE-035 ACGCCGCCGA 5051 TEE-036 GGCATTCATCGT5052 TEE-037 GCATTAGTATCT 5053 TEE-038 TCGGTTATTGTT 5054 TEE-039TCCAATTGGGAA 5055 TEE-040 ATCTATTGGCCA 5056 TEE-041 TTACTGGGTGTT 5057TEE-042 AGGGTGAAGGTC 5058 TEE-043 GGTGGGTGTGTC 5059 TEE-044 CGCTTCAATGCT5060 TEE-045 TGCTTCAATGCC 5061 TEE-046 TGTGTCTTTGCA 5062 TEE-047CACGGGGACAGC 5063 TEE-048 AAGCTGTACATG 5064 TEE-049 GATGGGGGCACA 5065TEE-050 ATATGTGCCCTT 5066 TEE-051 TCCTTCTGGGTC 5067 TEE-052 GGTGGGTGTGTC5068 TEE-053 GAATGGATGGGG 5069 TEE-054 CAXGTGATATTC 5070 TEE-055AGGAGGGTTTGT 5071 TEE-056 TGGGCGAGTGGG 5072 TEE-057 CGGCTCACCAGT 5073TEE-058 GGTTTCXATAAC 5074 TEE-059 GGTGGGTGTGTC 5075 TEE-060 TTACTGGGTGTT5076 TEE-061 AAGTCTTTGGGT 5077 TEE-062 CCGGCGGGU — TEE-063 CCGGCGGG —TEE-064 CCGGCGG — TEE-065 CCGGCG — TEE-066 CCGGC — TEE-067 CGGCGGGU —TEE-068 GGGAGACGGCGGCGGTGGCGGCGCGGGCAGAGCAA 5078GGACGCGGCGGATCCCACTCGCACAGCAGCGCACTC GGTGCCCCGCGCAGGGTCG TEE-069AAAGAAATGGAATCGAAGAGAATGGAAACAAATGG 5079AATGGAATTGAATGGAATGGAATTGAATGGAATGGG AACG TEE-070AAAGAAATGGAATCGAAGAGAATGGAAACAAATGG 5080AATGGAATTGAATGGAATGGAATTGAATGGAATGGG AACG TEE-071AGACAGTCAGACAATCACAAAGAAACAAGAATGAA 5081AATGAATGAACAAAACCTTCAAGAAATATGGGATTA TGAAGAGGCCAAATGT TEE-072AAAAGGAAATACAAGACAACAAACACAGAAACACA 5082ACCATCGGGCATCATGAAACCTCGTGAAGATAATCA TCAGGGT TEE-073AGACCCTAATATCACAGTTAAACGAACTAGAGAAGG 5083AAGAGCAAACAAATTCAAAAGCTAGCGGAAAGCAA GAAATAACTAAGACCAG TEE-074AAAGACTTAAACATAAGACCTAAAACCATAAAAACC 5084ACAGAAGAAAACATAGGCAATGCCATTCAGGACATA GGCATGGGCAAAGACTTC TEE-075AGCAATAACCAAACAACCTCATTAAAAAGTAGGCAA 5085AGGACATAAACAGACACTTTTCAAAAGAAGACATAC ACGTGGCCAACAAACATATG TEE-076AGAAAGAATCAAGAGGAAATGCAAGAAATCCAAAA 5086CACTGTAACAGATATGATGAATAATGAGGTATGCAC TCATCAGCAGACTCGACAT TEE-077GCACTAGTCAGATCAAGACAGAAAGTCAACGAACAA 5087AGAACAGACTTAAACTACACTCTAGAACAAATGGAC CTA TEE-078AGCAGCCAACAAGCATATGAAATAATGCTCCACAAC 5088ACTCATCATCAGAGAAATGCAAATCAAAACCAAAAT TEE-079AATATACGCAAATCAATAAATGTAATCCAGCATATA 5089AACAGTACTAAAGACAAAAACCACATGATTATCTCA ATAGATGCAGAAAAGGCC TEE-080ATGTACACAAATCAATAAATGCAGTCCAGCATATAA 5090ACAGAACCAAACACAAAAACCACATGATTATCTCAA TAGATGCAGAAAAGGCCTTT TEE-081TATACCACACAAATGCAAAAGATTATTAGCAACAAT 5091TATCAACAGCAATATGTCAACAAGTTGACAAACCTA GAGGACATGGAT TEE-082AAACACACAAAGCAACAAAAGAACGAAGCAACAAA 5092AGCATAGATTTATTGAAATGAAAGTACATTCTACAG AGTGGGGGCAGGCT TEE-083GAAATCATCATCAAACGGAATCGAATGGAATCATTG 5093AATGGAATGGAATGGAATCATCATGGAATGGAAACG TEE-084AACAGAATGGAATCAAATCGAATGAAATGGAATGG 5094AATAGAAAGGAATGGAATGAAATGGAATGGAAAGG ATTCGAATGGAATGCAATCG TEE-085TACAAAGAACTCAAACAAATCAGCAAGAACAAAAA 5095CAATCCCAACAAAATGTTGGACAAAGACATGAATAG ACAATTCTCGAAAGAAGATGTACAAATGGCTTEE-086 TGTTGAGAGAAATTAAACAAAGCACAGATAAATGGA 5096AAAACGTGTTCATAGATTGAAAGACTTCATGTTGTAT GGTGTC TEE-087AAACGATTGGACAGGAATGGAATCACCATCGAATGG 5097AAACGAATGGAATCTTCGAATGGAATTGAATGAAATTATTGAACGGAATCAAATAGAATCATCATTGAACAG AATCAAATTGGATCAT TEE-088AACAATAAACAAACTCCAACTAGACACAATAGTCAA 5098ATTGCTGAAAATGAAATATAAAGGAACAATCTCGAT GGTAGCCCAAGGA TEE-089AAATCAATAAATGTAATTCAGCATATAAACAGAACC 5099AAAGACAAAAACCACATGATTATCTCAATAGATGCA GAAAAGGCCTTT TEE-090GCTCAAGGAAATAAAATAGGACACAAAGAAATGGA 5100AAAACATTCCATACTCATGGATAGAAAGAATCAATA TCATGAAATGGCC TEE-091AACATACGCAAATCAATAAATGTAATCCAGCATATA 5101AACAGAACCAAAGACAAAAACCACATGATTATCTCA ATAGATGCAGAAAAGGCC TEE-092AACAATCACTAGTCCTTAAGTAAGAGACAACACCTT 5102TTGTCACACACAGTTTGTCCTAACTTTATCTTGGTAA TTGGGGAGACC TEE-093AGAAAACACACAGACAACAAAAAACACAGAACGAC 5103 AATGACAAAATGGCCAAGC TEE-094ACACAACAACCAAGAAACAACCCCATTAAGAAGTGG 5104GAAAAATACATGAATAAACACATCTCAAAAGAAGAC AAACAAGTGGCTAAC TEE-095ACAGCAGAAAACGAACATCAGAAAATCACTCTACAT 5105GATGCTTAAATACAGAGGGCAAGCAACCCAAGAGA AAACACCACTTCCTAAT TEE-096GAATAGAACAGAATGGAATCAAATCGAATGAAATG 5106GAATGGAATAGAAAGGAATGGAATGAAATGGAATG GAAAGGATTCGAATGGAATG TEE-097TAAGCAGAGAAAATATCAACACGAAAATAATGCAA 5107GGAGAAAAATACAGAACAATCCAAAATGTGGCC TEE-098GAACAATCAATGGAAGCAGAAACAAATAAACCAAG 5108GTGTGCATCAAGGAATACATTCACGCATGATGGCTG TATGAGTAAAATG TEE-099GATCAATAAATGTAATTCATCATATAAACAGAGAAC 5109TAAAGACAAAAACACATGATTATCGCAATACATGCA GAAAAGGCC TEE-100GACAAGAGTTCAGAAAGGAAGACTACACAGAAATA 5110CGCATTTTAAAGTCACTGACATGGAGATGACACTTA AAACCATGAACATGGATGGG TEE-101AAGCAAAGAAAGAATGAAGCAGCAAAAGAACGAAA 5111GCAGGAATTTATTGAAAACCAAAGTACACTCCACAG TATGGGAGCGGACCCGAGCA TEE-102ACCAACATAAGACAAAGAAACATCCAGCAGCTGCCT 5112ATGGCAAAAGATTACAATGTGTCAAACAAGAGGGCA ATG TEE-103GGACAAATTGCTAGAAATAAACAAATTACCAAAAAT 5113GATTCAAGTAGAGACAGAGAATCAAAATAGAACTAC ACATAAGTGGGCCAAG TEE-104AACATAATCCATCAAATAAACAGAACCAAAGACAAA 5114AACCACATGATTATCTCAATAGATGCAGAAAAGGCC TTC TEE-105AAAATCAATATGAAAACAAACACAAGCAGACAAAG 5115AAAATTGGGCAAAAGGTTTGAGCAGACACTTCACCA AAGAAGTACAAATGGCAAATCAGCA TEE-106AACCAAATTAGACAAATTGGAAATCATTACACATAA 5116CAAAAGTAATAAACTGTCAGCCTCAGTAGTATTCATT GTACATAAACTGGCC TEE-107AAGGAATTTAAGCAAATCAACAAGCAAAACCAAAAT 5117AATCCCATTAAAAAGTGGGTAAAGGACATGAATACA CACTTGTCAATAGAGGACATTCAAGTGGCCAACTEE-108 TAACCTGATTTGCCATAATCCACGATACGCTTACAAC 5118AGTGATATACAAGTTACATGAGAAACACAAACATTT TGCAAGGAAACTGTGGCCAGATG TEE-109AACTAACACAAGAACAGAAAACCAAACATCACATGT 5119TCTCACTCATAAGCGGGAGCTGAACAATGAGAACAC ACGGACACAGGGAGAGGAACATG TEE-110TAAACTGACACAAACACAGACACACAGATACACACA 5120TACATACAGAAATACACATTCACACACAGACCTGGT CTTTGGAGCCAGAGATG TEE-111ATCAACAGACAACAGAAACAAATCCACAAAGCACTT 5121AGTTATTAGAACTGTCATACAGACTGTACAACAACC ACATTTACCAT TEE-112AAATAAGCCAACGGTCATAAATTGCAAAGCCTTTTA 5122CAATCCAAACATGATGGAAACGATATGCCATTTTGA AGGTGATTTGAAAAGCACATGGTTT TEE-113AAACAGTTCAAAAATTATTGCAACAAAATGAGAGAG 5123ATGAGTTTATCTTGCAAACTAATGGATGGTAGCAGT GACAGTGGCAAAACGTGGTTTGATTCT TEE-114TAAGCAACTTCAGCAAAGTCTCAGGATACAAAATCA 5124ATGTACAAAAATCACAAGCATTCTTATACACCAACA ACAGACAAACAAGAGTGCCAAATCATG TEE-115AGCAAACAAACAAACAAACAAACAAACTATGACAG 5125GAACAAAACGTCACATATCAACATTAACAAAGAATGTAAACAGCCTAAATGCTTCACTTAAAAGTTATAGAC AGGGGCTGGGCATGGTGGCTCACGCC TEE-116GGAAATAACAGAGAACACAAACAAATGGGAAAACA 5126TTCCATGTTCATGGATAGGAAGAATCAATATTGTGA AAATGGCCATACT TEE-117AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAAT 5127GTACAAAAATCACAAGCATTCTTATACACCAATAAC AGACAAACAGAGAGCC TEE-118AGATAAGAATAAGGCAAACATAGTAATAGGGAGTTC 5128ATGAATAACACACGGAAAGAGAACTTACAGGGCTGT GATCAGGAAACG TEE-119AGGAAATAAAAGAAGACACAAACAAATGGAAGAAC 5129ATTCCATGCTTATGGATAGGGAGAATCAGTATCGTG AAAATGGCCATACT TEE-120AACATACGAAAATCAATAAACGTAATCCAGCATATA 5130AACAGAACCAAAGACAAAAACCACATGATTATCTCA ATAGATGCAGAAAAGGCCTTT TEE-121AATGGACTCGAATGAAATCATCATCAAACGGAATCG 5131AATGGAATCATTGAATGGAATGGAATGGAATCATCA TGGAATGGAAACG TEE-122AAGATTTAAACATAAGACCTAAAACGACAAAAATCC 5132TAGGAGAAAACCTAAGCAATACCATTCAGGACATAG GCATGGGCAAAGACTTCATG TEE-123TAATGAGAAGACACAGACAACACAAAGAATCACAG 5133AAACATGACACAGGTGACAAGAACAGGCAAGGACC TGCAGTGCACAGGAGCC TEE-124TAAACGTTAGACCTAAAACCATAAAAACCCTAGAAG 5134AAAACCTAGGCATTACCATTCAGGACATAGGCATGG GCAAGGAC TEE-125GAATTGAATTGAATGGAATGGAATGCAATGGAATCT 5135AATGAAACGGAAAGGAAAGGAATGGAATGGAATGG AATG TEE-126GTAATGGAATGGAATGGAAAGGAATCGAAACGAAA 5136GGAATGGAGACAGATGGAATGGAATGGAACAGAG TEE-127AGAGAAATGCAAATCAAAACCACAATGGAATACCAT 5137CTCACGCCAGTCAGAATGGCAATTATTAAAAAATCA CAACAATTAATGATGGCAAGGCTGTGG TEE-128AACATACACAAATCAATAAACGTAATCCAGCTTATA 5138AACAGAACCAAAGACAAAAACCACATGATTATCTCA ATAGATGCGGAAAAGGCC TEE-129TAAACAGAACCAAAGACAAAAATCACATGATTATCT 5139 CAATAGATGCAGAAAAGGCC TEE-130AATGGAATGCAATCGAATGGAATGGAATCGAACGGA 5140ATGGAATAAAATGGAAGAAAACTGGCAAGAAATGG AATCG TEE-131AGATAAAAAGAACAGCAGCCAAAATGACAAAAGCA 5141AAAAGCAAAATCGTGTTAGAGCCAGGTGTGGTGATG TGTGCT TEE-132AGGAAAGTTTTCAATATGAGAAAGATACAAACCAAC 5142AGAATAAGCAAACTGGATAAACAGAAAATACAGAG AGAGCCAAGG TEE-133GCAATCTCAGGATACAAAATCAATGTGCAAAAATCA 5143CAAGCATTCTCATACACCAATAACAGACAAACAGAG CCAAATCATG TEE-134AGCATTCATATCTTGCAGTGTTGGGAAAGAGTGAGA 5144GGTTGTGATGTCAAGAAGGATAGGTCAGAAGTGGAA GGTATGGGGGATTGTGCCTGCTGTCATGGCTTEE-135 AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAAT 5145GTGCAAAAATCACAAGCATTCTTATACACCAATAAC AGACAAACAGAGAGCC TEE-136AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAAT 5146GTGCAAAAATCACAAGCATTCTTATACACCAACAAC AGACAAACAGAGAGCC TEE-137TAAGCCGATAAGCAACTTCAGCAAAGTCTCAGGAGA 5147CAAAATCAATGTGCAAAAAATCACAAGCATTCTTAT ACACTAATAACAGACAAACAGAGAGCCAAATCATGTEE-138 AACGTGACATACATACAAAAAGTTTTTAGAGCAAGT 5148GAAATTTTAGCTGCTATATGTTAATTGGTGGTAATCCC TEE-139TACGCAAATCGATAAATGTAATCCAGCATATAAACA 5149GAACCAAAGACAAAAACCACATGATTATCTCAATAG ATGCAGAAAAGGCC TEE-140GCAATCGAATGGAATGGAATCGAACGGAATGGAATA 5150AAATGGAAGAAAACTGGCAAGAAATGGAATCG TEE-141TTGAATCGAATGGAATCGAATGGATTGGAAAGGAAT 5151AGAATGGAATGGAATGGAATTGACTCAAATGGAATG TEE-142TAAAGAAAAACAAACAAACAGAAATCAATGAAAAT 5152CCCATTCAAAGGTCAGCAACCTCAAAGACTGAAGGT AGATAAGCCCACAAGGATG TEE-143GTCATATTTGGGATTTATCATCTGTTTCTATTGTTGTT 5153GTTTTAGTACACACAAAGCCACAATAAATATTCTAG GCT TEE-144AAAAGTACAGAAGACAACAAAAAATGAGAGAGAGA 5154AAGATAACAGACTATAGCAGCATTGGTGATCAGAGC CACCAG TEE-145AACCCACAAAGACAACAGAAGAAAAGACAACAGTA 5155GACAAGGATGTCAACCACATTTTGGAAGAGACAAGT AATCAAACACATGGCA TEE-146AAAGACCGAAACAACAACAGAAACAGAAACAAACA 5156ACAATAAGAAAAAATGTTAAGCAAAACAAATGATTG CACAACTTACATGATTACTGAGTGTTCTAATGGTTEE-147 AATCAGTAAACGTAATACAGCATATAAACAGAACCA 5157AAGACAAAAACCACATGATTATCTCAATAGATGCAG AAAAGGCC TEE-148AAGCAACTTCAGCAAAGTCTCAGGACACAAAATCAA 5158TATGCGAAAATCACAAGCATTCCTATACACCAATAA TAGACAAACAGAGAGCCAAATCATG TEE-149AGCAACTTCAGCAAAATCTCAGGATACAAAATCAAT 5159GTACAAAAATCACAAGCATTCTTATACACCAACAAC AGACAAACAGAGAGCC TEE-150TAATGCAAACTAAAACGACAATGAGATATCAATACA 5160TAACTACCAGAAAGGCTAACAAAAAAACAGTCATAA CACACCAAAGGCTGATGAGTGAGGATGTGCAGTEE-151 AGCAACTTCAGCAAAGTCTCAGGATACAAAATCGAT 5161GTGCAAAAATCACAAGCATTCTTATACACCAATAAC AGGCAAACAGAGAGCC TEE-152GATATATAAACAAGAAAACAACTAATCACAACTCAA 5162TATCAAAGTGCAATGATGGTGCAAAATGCAAGTATG GTGGGGACAGAGAAAGGATGC TEE-153AAGACAGAACACTGAAACTCAACAGAGAAGTAACA 5163AGAACACCTAAGACAAGGAAGGAGAGGGAAGGCAG GCAG TEE-154TAAGACACATAGAAAACATAAAGCAAAATGGCAGA 5164TGTAAATGCAACCTATCAATCAAAACATTACGAATG GCTT TEE-155TGAAACAAATGATAATGAAAATACAACATACCAAAC 5165ATACGAGATACAGTAAAAGCAGTACTAAGATGCAAG TATATATTGCTACAAGTGCCTAC TEE-156AATGTAATCCAGCATATAAACAGAGCCAAAGACAAA 5166AACCACATGATTATCTCAATAGATGCAGAAAAAGCCTTTGACAAAATTCAACAACCCTTCATGCTAAAAACTC TCAATAAATTAGGTATTGATGGGACG TEE-157ACAAAATTGATAGACCACTAGCAAGACTAATAAAGA 5167AGAAAAGAGAGAAGAATCATTACCATTCAGGACATA GGCATGGGCAAGGAC TEE-158AAGGATTCGAATGGAATGCAATCGAATGGAATGGAA 5168TCGAACGGAATGGAATAAAATGGAAGAAAACTGGC AAGAAATGGAATCG TEE-159GATCATCAGAGAAACAGAGAAATGCAAATTAAAACC 5169ACAATGAGATACTATCTCCACACAAGTCAGAATGGC TAT TEE-160ATCAAAAGAAAAGCAACCTAACAAATACGGGAAGA 5170ATATTTGAATAGACATTTCACAGGAAAAGATATATG AATGGCCAAAAAGCAAATGAAAAG TEE-161AACAGCAATGACAATGATCAGTAACAACAAGACTTT 5171 TAACTTTGAAAAAATCAGGACC TEE-162AAGAGCCTGAATAGCTAAAGTGATCATAAGCAAAAA 5172GAACAAAGTCGGAAGCATCACATTACCTGACTTCAA ACTATACTCAAAGGCTATG TEE-163ACTCAGGAAAAATAACGAATCCAACTCACAGGAGAA 5173AGAAGTACAAACCAGAAACCAATTTCAAATTACAAG GACCAGAATACTCATGTTGGCTGGCCAGTTEE-164 TTGACCAGAACACATTACACAATGCTAATCAACTGC 5174AAAGGAGAATATGAACAGAGAGGAGGACATGGATA TTTTGTG TEE-165AACATATGGAAAAAAACTCAACATCACTGATCATTA 5175GAGAAATGCAAATCAAAACCACAATGAGATACCATC TCACGCCAGTCAGAATGGCG TEE-166AGCAACTTCAGCAAAGACTCAGGATACAAAATCAAT 5176GTGCAAAAATCACAAGCATTCTTATACACCAATAAC AGACAGAGAGCCAAAT TEE-167TGGGATATGGGTGAAAGAACAAGTTTGCAGAAAAGA 5177TACAGTGAATTATGGACCATGAGTTCGGGAAAGAAG GGTAGGACTGCG TEE-168AGCAGTGCAAGAACAACATAACATACAAGTAAACA 5178AACACATGGGGCCAGGTAATAAAAAGTCAGGCTCAA GAGGTCAG TEE-169AAGGAAAAGTAAAAGGAACTTAACACCTTCAAGAA 5179AAGACAGACAAATAACAAAACAGCAGTTTGATAGA ATGAGATATCAGGGGATGGCA TEE-170GCTAGTTCAACATATGCAAATCAATAAACGTAATCC 5180ATCACATAAACAGAACCAATGACAAAAACCACGATT ATCTCAATAGATGCAGAAAAGGCC TEE-171AACATCACTGATCATTAGAAACACACAAATCAAAAC 5181CACAATAAGATACCATCTAACACCAGTCACAATGGC TATT TEE-172AGAGCATCCACAAGGCCCAATTCAAAGAATCTGAAA 5182TAATGTATTGTTACTGCAACAGTTGTGAGTACCAGTG GCATCAG TEE-173GGAATAACAACAACAACAACCAAAAGACATATAGA 5183AAACAAACAGCACGATGGCAGATGTAAAGCCTACC TEE-174AAACGCAGAAACAAATCAACGAAAGAACGAAGCAA 5184TGAAAGACAAAGCAACAAAAGAATGGAGTAAGAAA GCACACTCCACAAAGTGGAAGCAGGCTGGGACATEE-175 AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAAT 5185GTGCAAAAATCACAAGCATTCCTATACACCAACAAC AGACAAACAGAGAGCC TEE-176AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAAT 5186GGGAAAAAATCACAAGCATTCCTATACATCAATAAC AGACAAACAGAGAGCC TEE-177ACACATTTCAAGGAAGGAAACAAGAACAGACAGAA 5187ACACAACATACTTCATGAAACCACATTTTAGCATCCT GGCCGAGTATTCATCA TEE-178AGCAACTTCAGCAAAGTCTCAGGACACAAAATCAAT 5188GTGCAAAAATCACAAGCATTCTTATACACCAATAAC AGACAAACAGAGAGCC TEE-179TATTTTACCAGATTATTCAAGCAATATATAGACAGCT 5189TAAAGCATACAAGAAGACATGTATAGATTTACATGCAAACACTGCACCACTTTACATAAGGGACTTGAGCAC TEE-180CCCAACTTCAAATTATACTACAAGGCTACAGTAATC 5190AAAAAAGCATAGTACTATTACAAAAACAGACACACA GGCCAATGGAATACAAT TEE-181AGAAAGGATTCGAATGGAATGAAAAAGAATTGAAT 5191GGAATAGAACAGAATGGAATCAAATCGAATGAAAT GGAATGGAATAGAAAGGAATGGAATG TEE-182GTTTACAGTCAAGTGTACAAACAGAATATAAGCAAA 5192CAAAAGAGAACATATACTTACAAACTATGCTAAGTG CCATGAAGGAAAAG TEE-183AAGAGTATTGAAGTTGACATATCTAGACTGATCAAG 5193AACAAAGACAAAAGGTACAGATTATCAAGAAAATG AGCGGGCAAAGCAAGATGGCC TEE-184AGTAGAATTGCAATTGCAAATTTCACACATATACTCA 5194CACACAAGTACACACATCCACTTTTACAACTAAAAA AACTAGCACCCAGGACAGGTGCAGTGGCTTEE-185 TGAATGCTATAGAGCAGTAAAAACAAATAAATGAAC 5195TACATTACAGCTACTTACAACCATATGAAAGAATATAACCATAACAATGATGAGTGGACAAAAGCTAAGTGTGAAAGAATGCATAGTGCTACAGCAGCCAACATTTAC AGC TEE-186GAATGGAATCAAATAGAATGGAATCGAAACAAATG 5196GAATGGAATGGAATGGGAGCTGAGATTGTGTCACTG CAC TEE-187TAAAAGTGTGCTCAACATCATTGATCATCAGAGAAA 5197TGCAAATCAAAACTACAATGAGATATCATCTCATCC CAGTCAAAGTGGCT TEE-188TCAGACCATAGCAGATAACATGCACATTAGCAATAC 5198 GATTGCCATGACAGAGTGGTTGGTGTEE-189 ACAAACAATCCAATTCGAAAATGGGCAAGATATTTC 5199ACCAAAGACATGAGCTGATATTTCAC TEE-190 AGGAAAAACAACAACAACAACAGGAAAACAACCTC5200 AGTATGAAGACAAGTACATTGATTTATTCAACATTTA CTGATCACTTTTCAGGTGGTAGGCAGTEE-191 AACAAAACAAAAACCCAACTCAATAACAAGAAGAC 5201AAACAACCCAATTTAAAATGAGCAAAGAACTTGATA AACATGTCTCCAAAGAAGATACGGCCAAAGAGCACTEE-192 ATACAACTAAAGCAAATATAAGCAACTAAAGCAACA 5202GTACAACTAAAGCAAAACAGAACAAGACTGCCAGG GCCTAGAAAAGCCAAGAAC TEE-193AACAACAACAACAACAGGAAAACAACCTCAGTATG 5203AAGACAAGTACATTGATTTATTCAACATTTACTGATC ACTTTTCAGGTGGTAGGCAGACC TEE-194AGAGAGTATTCATCATGAGGAGTATTACTGGACAAA 5204TAATTCACAAACGAACAAACCAAAGCGATCATCTTT GTACTGGCTGGCTA TEE-195AGTAAATCACCATAAAGAAGGTAAGAGTTCATTCAC 5205AAAAACAACAAACTGAAGAATCAGGCCATAGTA TEE-196AAAATAGAATGAAAGAGAATCAAATGGAATTGAATC 5206GAATGGAATCGAATGGATTGGAAAGGAATAGAATG GAATGGAATGGAATG TEE-197AAAAGATGCAAAAGTAGCAAATGCAATGTTAAAACA 5207AGCAAAGAAAGAATCAGGTGGACCACATAGTGCAGT GCTTCTC TEE-198TTCACAGCAGCATTACGCACAATAGCCAGAAGGTGG 5208 GAACAGACAAAATGCCTTTTGATGGGTEE-199 CCATAACACAATTAAAAACAACCTAAATGTCTAATA 5209GAAGAACACTGTTCAGACCGGGCATGGTGGCTTATA CC TEE-200TGGATTTCAGATATTTAACACAAAATAGTCAAAGCA 5210GATAAATACTAGCAACTTATTTTTAATGGGTAACATC ATATGTTCGTGCCTT TEE-201ATCATTGAATGCAATCACATGGAATCATCACAGAAT 5211GGAATCGTACGGAATCATCATCGAATGGAATTGAATGGAATCATCAATTGGACTCGAATGGAAACATCAAAT GGAATCGATTGGAAGTGTCGAATGGACTCGTEE-202 AGAAACAGCCAGAAAACAATTATTACCTACAGCATT 5212AAAACTATTCAAATGACAGCATATTTTTCAGCAGAA ATCATGAAGGCCAGAAGGACGTGTCAT TEE-203AAAATGATCATGAGAAAATTCAGCAACAAAACCATG 5213AAATTGCAAAGATATTACTTTTGGGATGGAACAGAG CTGGAAGGCAAAGAG TEE-204AACCACTGCTCAAGGAAATAAGAGAGAACACAAAC 5214AAATGAAAAAACATTCCATGCTCATGGATAGGAAGA ATCAG TEE-205TACTCTCAGAAGGGAAGCAGATATTCAGCATAAATC 5215ATATTGTTTGTACAAAGAGTCTGGGCATGGTGAATG ACACT TEE-206TATAGTTGAATGAACACACATACACACACACATGCC 5216ACAAAACAAAAACAAAGTTATCCTCACACACAGGATAGAAACCAAACCAAATCCCAACACATGGCAAGATGAT TEE-207GCTCAAAGAAATCAGAAATGACACAAGCAAATGGA 5217AAAACATGCCATGTTCATGAATATGAAGAATCAATA TTGTTAAAATGGCCATACTGCTCA TEE-208GGATACAAAATCAATGTACAAAAATCACAAGCATTC 5218TTATACACCAATAACAGACAAACAGAGAGCC TEE-209AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAAT 5219GTACAAAAATCACAAGCATTCTTATACACCAACAAC AGACAAACAGAGAGCC TEE-210AGGAGAATAGCAGTAGAATGACAAAATTAGATTTTC 5220ACATGAAACTTGATGACAGTGTAGGAAATGGACTGA AAGGACAAGAC TEE-211AGCAACTTCAGCAAAGTCTCGGGATACAAAATCAAT 5221GTGCAAAAATCACAAGCATTCCTATACACCAATAAC AGGCAAACAGAGAGCC TEE-212AAGTTCAAACATCAGTATTAACCTTGAACATCAATG 5222GCCTACATGCATCACTTAAAACATACAGACAGGCAA ATTGGGTTAAGAAAACAAACAAGCAAACAAAACATGTTCCAAACATTTGTTGGCTAT TEE-213 AAGAAACAATCAAAAGGAAGTGCTAGAAATAAAAC 5223ACACTGTAATAGAAAAGAAGAATGCCTTATGGGCTT ATCAATAGACTAGACATGGCCAGG TEE-214AAAGAAAGACAGAGAACAAACGTAATTCAAGATGA 5224CTGATTACATATCCAAGAACATTAGATGGTCAAAGACTTTAAGAAGGAATACATTCAAAGGCAAAAAGTCAC TTACTGATTTTGGTGGAGTTTGCCACATGGACTEE-215 AGCAACTTCAGCAAAGTTTCAGGATACAAAATCAAT 5225GTGCAAAAATCACAAGCATTCTTATACACCAACAAC AGACAAACAGAGAGCC TEE-216AGAATCAAATGGAATTGAATCGAATGGAATCGAATG 5226GATTGGAAAGGAATAGAATGGAATGGAATGGAATG TEE-217AAACAGAACCACAGATATCTGTAAAGGATTACACTA 5227TAGTATTCAACAGAGTATGGAACAGAGTATAGTATT CAACAGAGTATGCAAAGAAACTAAGGCCAGAAAGTEE-218 AAAAAATGTTCAACATCACTAGTCAGCAGAGAAATG 5228CAAATCAAAATCACAATGAGATAACTTCTCACACCA GACAGCATGGC TEE-219GAATCCATGTTCATAGCACAACAACCAAACAGAAGA 5229AATCACTGTGAAATAAGAAACAAAGCAAAACACAG ATGTCGACACATGGCA TEE-220AGGATACAAAATCAAAGTGCAAAAATCACAAGCATT 5230CTTATACACCAATAACAGACAAACAGAGAGCC TEE-221AACAGATTTAAACAAACCAACAAGCAAAAAACGAA 5231CAACTCCATTCAAACATGGACAAAAGACACGAACAG ACACTTTTCAAAGAAGACATACATGTGGCCTEE-222 AAAGACAATATACAAATGGCCAATAAGCACATGAAA 5232AGACGCTCAACATCCTTAGTCGTTAAGGCAATGCAA ATCAAAACCACAATG TEE-223TAAACAACGAGAACACATGAACACAAAGAGGGGAA 5233CAACAGACACCAAGACCTTCTTGAGGGTGGAGGATG GGAGGAGGGAG TEE-224GGTTCAACTTACAATATTTTGACTTGACAACAGTGCA 5234AAAGCAATACACGATTAGTAGAAACACACTTCCAAT GCCCATAGGACCATTCTGC TEE-225AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAAT 5235GAGCAAAAATCACAAGCATTCTTACACACCAATAAC AGACAAACAGAGAGCC TEE-226AATCCAGCATATAAACAGAACCAAAGACAAAAACC 5236ACATGATTATCTCAATAGATGCAGAAAAGGCC TEE-227TGAAAATACAAATGACCATGCAAGTAATTCCGCAGG 5237GAGAGAGCGGATATGAACAAACAGAAGAAATCAGA TGGGATAGTGCTGGCGGGAAGTCA TEE-228GCAAATGATTATAAGTGCTGTTATAGAAACATTCAA 5238AGACCAGAAAAGGACCACAATGGCTGACCAC TEE-229AGTCAATAACAAGAAGACAAACAACCCAATTACAAA 5239ATGGGATATGAATTTAATAGATGTTACTCCAAGGAA GATACACAAATGGCCAAC TEE-230ATGGTTAAAACTCAACAATGAAAACACAAACAGCGC 5240AATTTAAAAATGGGCAAAATGACAGGCCAGACCCAG TGGCTCATGCG TEE-231TAACTACTCACAGAACTCAACAAAACACTATACATG 5241CATTTACCAGTTTATTATAAAGATACAAGTCAGGAA CAGCCAAATGGAAGAAATGTAAATGGCAAGTEE-232 AACAGACCATAAATAAACACAGAAGACACACGAGT 5242GTAAAGTCAGTGCCCCGCTGCGAATTAAATCGGGGT GATGTGATGGCGAGTGAGTGGGTAGTT TEE-233GAATAGAATAGAATGGAATCATCGAATGGAATCGAA 5243TGGAATCATCATGATATGGAATTGAGTGGAATC TEE-234GGAATCTATAATACAGCTGTTTATAGCCAAGCACTA 5244AATCATATGATACAGAAAACAAATGCAGATGGTTTG AAGGGTGGG TEE-235AAGATAGAGTTGAAACAGTGGACAATTAAAGAGTAA 5245TTTGGAAGAATGGTGAAATTACAGCCATGCTTTGAA TCAGGCGGGTTCACTGGC TEE-236TGAAAAGAAGAATGACCATAAGCAAGCAGATGAAA 5246AACAAAACAGAATTTTTACAGACGTCTTGGACTGAT ATCTTGGGC TEE-237AGGAATCTATAATACAGCTGTTTATAGCCAAGCACT 5247AAATCATATGATACAGAAAACAAATGCAGATGGTTT GAAGGGTGGG TEE-238AGGAAAAGAAAGAAATAGAAAATGCGAAATGGTAA 5248GAAAAAACAGCATAATAAACATTTGTATGGTGTTGA TGGACAATGCATT TEE-239TAACAGTACCAAAAAACAGTCATAATCTTCAAGAGC 5249TTAAATTTAGCATGAAAGGAAGACATTCATCAAAGAATCACACAAAGGAATGTAAAATTAAATGGAGATTAG TGCCAGGAAAGAGC TEE-240GCAAAACACAAACAACGCCATAAAAAACTGGGCAA 5250AGGATATGAACAGACATTTTTCAAAACAAAACATAC TTATGGCCAAC TEE-241AACAAAATTGAACAACATGCAAAGAAACATAAACG 5251AAGCAATGAAAGTGTGCAGATCCACTGAAATGAAAG TGCTGTCCAGAGTGGGAGCCAGCTCGAGATEE-242 GAATGGAATCAACATCAAACGGAATCAAACGGAATT 5252ATCGAATGGAATCGAAGAGAATCATCGAATGGCCACGAATGGAATCATCTAATGGAATGGAATGGAATAATC CATGG TEE-243TACAAGAAAATCACAGTAACATTTATAAAACACAGA 5253AGTGTGAACACACAGCTATTGACCTTGAAAACAGTG AAAGAGGGTCAGCTGTAGAACTAAGACATAAGCAAAGTTTTTCAATCAAGAATACATGGGTGGCC TEE-244AAGAATTGGACAAAACACACAAACAAAGCAAGGAA 5254GGAATGAAAGGATTTGTTGAAAATGAAAGTACACTC CACAGTGTGGGAGCAG TEE-245ACAGTTAACAAAAACCGAACAATCTAATTACGAAAT 5255GAACAAAAGATATGAACAGACATTTCACCCGAGAGT ATACAGGGGCCAGGCATGGT TEE-246AAACGCACAAACAAAGCAAGGAAAGAATGAAGCAA 5256CAAAAGCAGAGATTTATTGAAAATGAAAAATACACT CCACAGGGTGGG TEE-247CACCATGAGTCATTAGGTAAATGCAAATCAAAACCA 5257CAATGAAATACTTCACACCCATGAAGATGGCTATAA TAAAAAAACAGACA TEE-248AGCAACTTCAGCAAAGTCTCAGGAGACAAAATCAAT 5258GTACAAAAATCACAAGCATTCTTATACACCAATAAC AGACAAACAGAGAGCC TEE-249TGACATGCAAGAAATAAGGAAGTGCAAAAACAAAC 5259AAACAAACAACAACAACAACAACAACAACAACAAC AAAAAACAGTCCCAAAAGGATGGGCAG TEE-250AGACTTGAAAAGCACAGACAACGAAAGCAAAAATG 5260GACAAATGGAATCACATCAAGCTAAAAGGTTTTGCA TGGCAAAGG TEE-251GCAAAAGAAACAATCAGTAGAGTAAACAGACAACT 5261CATAGAATGCAAGAAAATCATCGCAATCTGTACATC CAACAAAGGGCT TEE-252ACAAAATCAAACTAACCTCGATAAGAATGCAAGTGA 5262ATCAAAATGAGTTTCAAGGGGTTGTGGCTAGTACAC GCTTTCTACAGCTG TEE-253ACAAACCACTGCTCAAGGAAATAAGGACACAAACA 5263AATGGAACAACATTCCGTGCTCATGGATAGGAAGAA TCAATATCGTGAAAATGGCCATACT TEE-254GAACGATTTATCACTGAAAATTAATACTCATGCAAG 5264TAGTAAACGAATGTAATGACCATGATAAGGAGACGG ACGGTGGTGATAGT TEE-255AGCAGAAGAAATAACTGAAATCAGAGTGAAACTGA 5265ATCAAATTGAGATGCAAAAATACATACGAAATGGCC AG TEE-256TGAATAGACACACAGACCAATGGAACAGAATAGAG 5266AACACAGAATAAATCTGCACACTTATAGCCAGCTGA TTTTTGACAAATTTGCCAAG TEE-257AGCAACTTCAGCAGTCTCAGTATACAAAAACAATGT 5267GCAAAAATCACAAGCATTCCTATATGCCAATAACAG ACAAACAGAGAGCC TEE-258ACCAATCAAGAAAACAATGCAACCCACAGAGAATG 5268 GACAAAAGCAAGGCAGGACAATGGCTTEE-259 GCCACAATTTTGAAACAACCATAATAATGAGAATAC 5269ACAAGACAACTCCAATAATGTGGGAAGACAAACTTT GCAATTCACATCATGGC TEE-260GAAAATGAACAATATGAACAAACAAACAAAATTACT 5270ACCCTTACGAAAGTACGTGCATTCTAGTATGGTGAC AAAAAGGAAA TEE-261TATGCAAATCAATAAACATAATCCATCACATAAACA 5271GAAACAAAGACAAAATGACATGATTATCTCAATAGA TGCAGAAAAGGCC TEE-262CACCCATCTGTAGGACCAGGAAGCCTGATGTGGGAG 5272AGAACAGCAGGCTAAATCCAGGGTTGGTCTCTACAGCAGAGGGAATCACAAGCCTGTTAGCAAGTGAAGAAC CAACACTGGCAAGAGTGTGAAGGCC TEE-263AGGATACAAAATCAATGTACAAAAATCACAAACATT 5273CTTATACACCAACAACAGACAAACAGAGAGCCAAAT CATGGGTG TEE-264AGGAAAATGCAAATCAGAACGACTATAACACACCAT 5274CTCAAACTCGTTAGGATGGCTATTATCAAAAAGTCA AGAGATAACAAATGTGGGCAAGGG TEE-265GTAACAAAACAGACTCATAGACCAATAGAACAGAAT 5275AGAGAATTCAGAAATAAGACTGCACTTCTATGACCA TGTGATCTTAGACAAACCT TEE-266AAAGGAAAACTACAAAACACTGCTGAAAGAAATCAT 5276TGACAACACAAACAAATGGAAACACATCCCAAGATC ATGGGTGGGTGGAATCAAT TEE-267ACACACATACCAACAGAACATGACAAAAGAACAAA 5277ACCAGCCGCATGCATACTCGATGGAGACAAAGGTAACACTGCAGAATGGTGAAGGAAGAACAGTCATTTTAA TGACAGTGTTGGCT TEE-268AACTAAGACAACAGATTGATTTACACTACTATTTTCA 5278CACAGCCAAAAATATCACTATGGCAATCGTCAAAAG GTCAATTCAAAGATGGGACAGT TEE-269GATCAGCTTAGAATACAATGGAACAGAACAGATTAG 5279AACAATGTGATTTTATTAGGGGCCACAGCACTGTTGACTCAAGTACAAGTTCTGACTCATGTAGAACTAACA CTTTT TEE-270GAATGGAATCAAATCGAATGAAATGGAATGGAATAG 5280AAAGGAATGGAATGAAATGGAATGGAAAGGATTCG AAT TEE-271AAATGAACAAAACTAGAGGAATGACATTACCTGACT 5281TCAAATTATACTACAGAGCTATAGTAACCAAAACAG CATGGTACAGGCAT TEE-272GGACAACATACACAAATCAGTCAAGATACATCATTT 5282CAACAGAATGAAAGACAAAAACCATTTGATCACTTC AATCGATGATGAAAAAGCA TEE-273AACTTCAGCAAATTCTCAGGATACAAAATCAATGTG 5283CAAAAACCACAAGCATTCCTATACACCAATAATAGA CAGTGAGCCAAAT TEE-274TATGACTTTCACAAATTACAGAAAAAGACACCCATT 5284TGACAAGGGAACTGAAGGTGGTGAAGACATACTGGC AGGCTAC TEE-275AACAGCAATAGACACAAAGTCAGCACTTACAGTACA 5285AAAACTAATGGCAAAAGCACATGAAGTGGGACAT TEE-276TGTAACACTGCAAACCATAAAAACCGTAGAAGAAAA 5286CCTAGACAATACTATTCAGGACATAGGCATGGGCAA AGAC TEE-277GAAGAAGAAAAAACATGGATATACAATGTCAACAG 5287AAATCAAGGAGAAACGGAATTTCACCAATCAATTTA GTGATCTGGGTT TEE-278AAAACACACAAACATACATGTGGATGCACATATAAA 5288CATGCACATACACACACACATAAATGCACAAACACA CTTAACACAAGCACACATGCAAACAAACACATGGTEE-279 TAGAAGGAATTTGATACATGCTCAGAAATACAGGCA 5289AAGGAAGTAGGTGCCTGCCAGTGAACACAGGGGAA CTATGGCTCCTA TEE-280TGACTAAACAGAGTTGAACAAGAACAAAAAGCAAA 5290TTTGCAGAAATGAAATACATACTAATTGAAAGTCCATGGACAGGCTCAACAGATGATATAGATACAGCTAAA GAGATAATTAGTGAAATGGATCAG TEE-281AAGTAATAAGACTGAATTAGTAATACAAAGTGTCTC 5291AACAAAGAAAATTGCGGGACTGTTCATGCTCATGGA CAGGAAGAATCAATATCATGAAAATGGCCTEE-282 ACAGACAGAGATTTAAAACAATAAACAAGCAGTAA 5292GCAAACACAGATAACAAAATGACATGATCCAACAAA TACTCAGAAGGAGACTTAGAAATGAATTGAGGGTCTEE-283 AGAAAAAAACAAACAGCCCATTAAAAGGTAGACAA 5293AGGACATGAACACTTTTCAAAAGAAGACATACATGT GGCCAAACAGCATG TEE-284AAAAATGACCAGAGCAATAGAATGCATTGACCAGAT 5294AAAGACCTTCACGTATGTTGAACTAAAATGTGTGGT GCAGGTG TEE-285AATCAGTCTAGATCTTAAAGGAACACCAGAGGGAGT 5295ATTTAAATGTGCCCAATAAGCAAGAATTATGGTGAT GTGGAAGTA TEE-286GAATGGAATGGAAAGGAATCGAAACGAAAGGAATG 5296GAGACAGATGGAATGGAATGGAACAGAGAGCAATGG TEE-287GGAATGGAATGAACACGAATGTAATGCAACCCAATA 5297GAATGGAATCGAATGGCATGGAATATAAAGAAATGG AATCGAAGAGAATGGAAACAAATGGAATGGAATTGTEE-288 AGGACATGAATAGACAATTCTCAAAAGAAGATACAC 5298AAGTGGCAAACAAACACATGAAAAAAGACTCAACA TTAGTAATGACCATGGAAATGCAAATC TEE-289TCCAGTCGATCATCATATAGTCAGCACTTATCATACA 5299CCAAGCCGTGTGCAAGGAAAGGGAATACAACCATGA ACATGATAGATGGATGGTT TEE-290TACAGATAAGAAAATTGAGACTCAAGAGTATTACAT 5300AAATTGTTTCAGCTACCACAGCAAAAAATGGTATGG TTGGGAATCAAGCTCAGGG TEE-291AGCCTATCAAAAAGTGGGCTAAGAATATGAATACAC 5301AATTCTCAAAAGAAGATATACAAATGGGCAACAAACATATGAAAACATACTCAACATCACTAATGATCAGGG AAATG TEE-292GAAAATGAACAATATGAACAAACAAACAAAATTACT 5302ACCCTTACGAAAGTACGTGCATTCTAGTATGGTGAC AAAAAGGAAAG TEE-293ACATACGCAAATCAATAAACATAATCCATCACATAA 5303ACAGAACCAAAGACAAAAATCACATGATTATCTCAA TAGATGCAGAAAAGGCCTTCGAC TEE-294AAGAGTATCAACAGTAAATTACATTAGCAGAAGAAT 5304CAACAAACATGAAAATAGAAATTATGGTAGCCAAAG AACAG TEE-295AATCGAATGGAATCAACATCAAACGGAAAAAAACG 5305GAATTATCGAATGGAATCGAAGAGAATCATCGAATG GACC TEE-296GAAAGGAATAGAATGGAATGGATCGTTATGGAAAG 5306ACATCGAATGGGATGGAATTGACTCGAATGGATTGGACTGGAATGGAACGGACTCGAATGGAATGGACTGGA ATG TEE-297TAAGCAATTTCAGCAGTCTCAGGATACAAAATCAAT 5307GTGCAAAAATCACAAGCATTCTTATACACCAACAAC AGACAAACAGAGAGCCAAATCG TEE-298AACGGAATCAAACGGAATTATCGAATGGAATCGAAG 5308AGAATCATCGAATGGCCACGAATGGAATCATCTAAT GGAATGGAATGGAATAATCCATGGACCCGAATGTEE-299 ACATCAAACGGAATCAAACGGAATTATCGAATGGAA 5309TCGAAAAGAATCATCGAACGGACTCGAATGGAATCA TCTAATGGAATGGAATGGAAG TEE-300ATCGAATGGAATCAACATCAAACGGAAAAAAACGG 5310AATTATCAAATGGAATCGAAGAGAATCATCGAATGG ACC TEE-301GAATAATCATTGAACGGAATCGAATGGAAACATCAT 5311CGAATGGAAACGAATGGAATCATCATCGAATGGAAA TGAAAGGAGTCATC TEE-302CATCAAACGGAATCAAACGGAATTATCGAATGGAAT 5312CGAAAAGAATCATCGAACGGACTCGAATGGAATCATCTAATGGAATGGAATGGAAGAATCCATGGACTCGAA TG TEE-303AAACGGAATCAAACGGAATTATCGAATGGAATCGAA 5313GAGAATCATCGAATGGACTCGAATGGAATCATCTAA TGGAATGGAATGGAAGAATCCATGG TEE-304ATACACAAATCAATAAATGTAATCCAGCATATAAAC 5314AGAACCAAAGACAAAAACCATATGATTATCTCAATG GATGCAGAAAAGGCC TEE-305AATCGAATAGAATCATCGAATGGACTCGAATGGAAT 5315 CATCGAATGTAATGATGGAACAGTCTEE-306 TGGAATGGAATCATCGCATAGAATCGAATGGAATTA 5316CCATCGAATGGGATCGAATGGTATCAACATCAAACG CAAAAAAACGGAATTATCGAATGGAATCGAAGAGAATCTTCGAACGGACCCG TEE-307 ATGGAATGGAATGGAATGGAATTAAATGGAATGGAA 5317AGGAATGGAATCGAATGGAAAGGAATC TEE-308 GTCGAAATGAATAGAATGCAATCATCATCAAATGGA5318 ATCCAATGGAATCATCATCAAATAGAATCGAATGGA ATCATCAAATGGAATCGAATGGAGTCATTGTEE-309 TGGAATTATCGAAAGCAAACGAATAGAATCATCGAA 5319TGGACTCGAATGGAATCATCGAATGGAATGGAATGG AACAG TEE-310AAAGGAATGGAATGCAATGGAATGCAATGGAATGC 5320ACAGGAATGGAATGGAATGGAATGGAAAGGAATG TEE-311AATCTAATGGAATCAACATCAAACGGAAAAAAACGG 5321AATTATCGAATGGAATCGAAGAGAATCATCGAATGG ACC TEE-312TACACAACAAAAGAAATACTCAACACAGTAAACAGA 5322CAACCTTCAGAACAGGAGAAAATATTTGCAAATACA TCTAACAAAGGGCTAATATCCAGAATCTTEE-313 TGCAATCCTAGTCTCAGATAAAACAGACATTAAACC 5323AACAAAGATCAAAAGAGACAAAGAAGGCCATTAC TEE-314GAATCGAATGGAATCAACATCAAACGGAAAAAAAC 5324GGAATTATCGAATGGAATCGAAAAGAATCATCGAAT GGACC TEE-315AATGGAATCGAATGGAATGCAATCCAATGGAATGGA 5325ATGCAATGCAATGGAATGGAATCGAACGGAATGCAG TGGAAGGGAATGG TEE-316GAACACAGAAAAATTTCAAAGGAATAATCAACAGG 5326GATTGATAACTAACTGGATTTAGAGAGCCAAGGCAA AGAGAATCAAAGCACAGGGCCTGAGTCGGAGTEE-317 AGTTGAATAGAACCAATCCGAATGAAATGGAATGGA 5327ATGGAACGGAATGGAATTGAATGGAATGGAATGGA ATGCAATGGA TEE-318AACTCGATTGCAATGGAATGTAATGTAATGGAATGG 5328AATGGAATTAAC GC GAATAGAATGGAAT GGAATGTA ATGGAACGGAATGGAATG TEE-319AAGCGGAATAGAATTGAATCATCATTGAATGGAATC 5329GAGTAGAATCATTGAAATCGAATGGAATCATAGAAT GGAATCCAAT TEE-320AATGGAATCGAAAGGAATAGAATGGAATGGATCGTT 5330ATGGAAAGATATCGAATGGAATGGAATTGACTCGAA TGGAATGGACTGGAATGGAACG TEE-321TAACGGAATAATCATCGAACAGAATCAAATGGAATC 5331ATCATTGAATGGAATTGAATGGAATCTTCGAATAGA CATGAATGGACCATCATCG TEE-322AACGGAATCAAACGGAATTATCGAATGGAATCGAAT 5332AGAATCATCGAACGGACTCGAATGGAATCATCTAAT GGAATGGAATGGAAG TEE-323ATTGGAATGGAACGGAACAGAACGGAATGGAATGG 5333AATAGAATGGAATGGAATGGAATGGTATGGAATGGA ATGGAATGGTACG TEE-324AATCCACAAAGACAACAGAAGAAAAGACAACAGTA 5334GACAAGGATGTCAACCACATTTTGGAAGAGACAAGT AATCAAACACATGGCA TEE-325GAATCGAATGGAATCAACATCAAACGGAAAAAAAC 5335GGAATTATCGAATGGAATCGAAAAGAATCATCGAACGGACTCGAATGGAATCATCTAATGGAATGGAATGGA AGAATCCATGG TEE-326AATGGAATCGAATGGAATCATCATCAAATGGAATCT 5336AATGGAATCATTGAACGGAATTGGATGGAATCGTCAT TEE-327CAACATCAAACGGAAAAAAACGGAATTATCGAATGG 5337 AATCGAAGAGAATCATCGAATGGACCTEE-328 CACAACCAAAGCAATGAAAGAAAAGCACAGACTTAT 5338TGAAATGAAAGTACACACCACAGAATGGGAGCAGG CTCAAGCAAGC TEE-329ATCAAAGGGAATCAAGCGGAATTATCGAATGGAATC 5339GAAGAGAATCATCGAATGGACTCGAATGGAATCATG TGATGGAATGGAATGGAATAATCCACGGACTTEE-330 GGAATCGAATGGAATCAATATCAAACGGAGAAAAA 5340CGGAATTATCGAATGGAATCGAAGAGAATCATCGAA TGGACC TEE-331AGGAATGGACACGAACGGAATGCAATCGAATGGAA 5341TGGAATCTAATAGAAAGGAATTGAATGAAATGGACT GG TEE-332GGAAGGGAATCAAATGCAACAGAATGTAATGGAAT 5342GGAATGCAATGGAATGCAATGGAATGGAATGGAATG CAATGGAATGG TEE-333AAATTGGATTGAATCGAATCGAATGGAAAAAATGAA 5343ATCAAATGAAATTGAATGGAATCGAAATGAATGTAAACAATGGAATCCAATGGAATCCAATGGAATCGAATC AAATGGTTTTGAGTGGCGTAAAATG TEE-334AATGGAAGGGAATGGAATGGAATCGAATCGAATGG 5344AACAGAATTCAATGGAATGGAATGGAATGGAATGGA ATCGAATGGAATGG TEE-335GAAAAATCATTGAACGGAATCGAATGGAATCATCAT 5345CGGATGGAAACGAATGGAATCATCATCGAATGGAAA TGAAAGGAGTCATC TEE-336GGAATCGAATGGAATCAACATCAAACGGAGAAAAA 5346CGGAATTATCGAATGGAATCGAAGAGAATCATCGAA TGGACC TEE-337AAAGAAATGTCACTGCGTATACACACACACGCACAT 5347ACACACACCATGGAATACTACTCAGCTATACAAAGG AATGAAATAATC CACAGC CAC TEE-338GGAATCGAATGGAATCAATATCAAACGGAAAAAAA 5348CGGAATTATCGAATGGAATCGAAGAGAATCATCGAA TGGACC TEE-339TGAACGGAATCGAATGGAATCATCATCGGATGGAAA 5349CGAATGGAATCATCATCGAATGGAAATGAAAGGAGT CATC TEE-340GAATAGAACGAAATGGAATGGAATGGAATGGAATG 5350 GAAAGGAATGGAATGGAATGGAACGTEE-341 TGGAATTATCGTCGAATAGAATCGAATGGTATCAAC 5351ATCAAACGGAAAAAAACGGAATTATCGAATGGAATCGAAGAGAATCATCGAACGGACTCGAATGGAATCATC TAATGGAATGGAATGGAATAATCCATGGTEE-342 GACAAAAAGAATCATCATCGAATAGAATCAAATGGA 5352ATCTTTGAATGGACTCAAAAGGAATATCGTCAAATGGAATCAAAAGCCATCATCGAATGGACTGAAATGGAA TTATCAAATGGACTCG TEE-343AACCAAACCAAGCAAACAAACAAACAGTAAAAACT 5353CAATAACAACCAACAAACAGGAAATACCAGGTAATT CAGATTATCTAGTTATGTGCCATAGT TEE-344GAATGAATTGAATGCAAACATCGAATGGTCTCGAAT 5354GGAATCATCTTCAAATGGAATGGAATGGAATCATCGCATAGAATCGAATGGAATTATCAACGAATGGAATCGAATGGAATCATCATCAGATGGAAATGAATGGAATCG TCAT TEE-345TGGAATGGAATCAAATCGCATGGAATCGAATGGAAT 5355AGAAAAGAATCAAACAGAGTGGAATGGAATGGAAT GGAATGGAATCATGCCGAATGGAATG TEE-346AAATGGAATAATGAAATGGAATCGAACGGAATCATC 5356ATCAAAAGGAACCGAATGAAGTCATTGAATGGAATCAAAGGCAATCATGGTCGAATGGAATCAAATGGAAACAGCATTGAATAGAATTGAATGGAGTCATCACATGGA ATCG TEE-347GAATTAACCCGAATAGAATGGAATGGAATGGAATGG 5357AACAGAACGGAACGGAATGGAATGGAATGGAATGG AATGGAATG TEE-348AAGATATACAAGCAGCCAACAAACATACGAAAGAA 5358TGCTCAACATCACTAATCCTCAGAGAAATTTAAATCAAAACCACAATGAGTTACAATCTCATACCAGTCAGAAT TEE-349AGATAAGTGGATGAACAGATGGACAGATGGATGGAT 5359GGATGGATGGATGGATGGATGCCTGGAAGAAAGAA GAATGGATAGTAAGCTGGGTATA TEE-350AGAATTACAAACCACTGCTCAACAAAATAAAAGAGT 5360ACACAAACAAATGGAAGAATATTCCATGCTTATGGA TAGGAAGAATCAATATTGTGAAAATGGCCATACTTEE-351 CATCGAATGGACTCGAATGGAATAATCATTGAACGG 5361AATCGAAGGGAATCATCATCGGATGGAAACGAATGG AATCATCATCGAATGGAAATG TEE-352AAAGGAATCAAACGGAATTATCGAATGGAATCGAAA 5362AGAATCATCGAACGGACTCGAATGGAATCATCTAAT GGAATGGAATGGAAGAATCCATGGACTCGAATGTEE-353 GGATATAAACAAGAAAACAACTAATCACAACTCAAT 5363ATCAAAGTGCAATGATGGTGCAAAATGCAAGTATGG TGGGGACAGAGAAAGGATGC TEE-354AACATCAAACGGAAAAAAACGGAAATATCGAATGG 5364 AATCGAAGAGAATCATCGAATGGACCTEE-355 TAAAATGGAATCGAATGGAATCAACATCAAATGGAA 5365TCAAATGGAATCATTGAACGGAATTGAATGGAATCG TCAT TEE-356AATCATCATCGAATGGAATCGAATGGTATCATTGAA 5366TGGAATCGAATGGAATCATCATCAGATGGAAATGAA TGGAATCGTCAT TEE-357CAATGCGTCAAGCTCAGACGTGCCTCACTACGGCAA 5367 TGCGTCAAGCTCAGGCGTGCCTCACTATTEE-358 TAAGCTGATAAGCAACTTTAGCAAAGTCTCAGGATA 5368CAAAATCAATGTACAAAAATCACAAGCATTCTTATA CACCAACAACAGACAGACGGAGAGCCAAATEE-359 AATCAAAGAATTGAATCGAATGGAATCATCTAATGT 5369 ACTCGAATGGAATCACCATTEE-360 ATGAACACGAATGTAATGCAATCCAATAGAATGGAA 5370TCGAATGGCATGGAATATAAAGAAATGGAATCGAAG AGAATGGAAACAAATGGAATGGAATTGAATGGAATGGAATTG TEE-361 ATCAAACGGAATCAAACGGAATTATCGAATGGAATC 5371GAAGAGAATCATCGAACGGACTCGAATGGAATCATC TAATGGAATGGGATGG TEE-362AATGGAAAGGAATCAAATGGAATATAATGGAATGCA 5372ATGGACTCGAATGGAATGGAATGGAATGGACCCAAA TGGAATGGAATGGAATGGAATG TEE-363GGAATACAACGGAATGGAATCGAAAAAAATGGAAA 5373GGAATGAAATGAATGGAATGGAATGGAATGGAATG GATGGGAATGGAATGGAATGG TEE-364GAATCAAGCGGAATTATCGAATGGAATCGAAGAGAA 5374TCATCGAAAGGACTCGAATGGAATCATCTAATGGAA TGGAATGGAATAATACACGGACC TEE-365AAGATAACCTGTGCCCAGGAGAAAAACAATCAATGG 5375CAACAAAAGCAGAAACAACACAAATGATACAATTA GCAGACAGAAACATTGAGATTGCTATT TEE-366AATGGACTCCAATGGAATAATCATTGAACGGAATCT 5376AATGGAATCATCATCGGATGGAAATGAGTGGAATCA TCATCGAATGGAATCG TEE-367AATCTATAAACGTAATCCATCACATAAACAGGACCA 5377AAGAGAAAAACCGCATGATTATCTCAAGAATGCAGA AAAGGCC TEE-368TAATTGATTCGAAATTAATGGAATTGAATGGAATGC 5378AATCAAATGGAATGGAATGTAATGCAATGGAATGTA ATAGAATGGAAAGCAATGGAATG TEE-369AAAGGAATGGACTTGAACAAAATGAAATCGAACGAT 5379AGGAATCGTACAGAACGGAAAGAAATGGAACGGAA TGGAATG TEE-370TGAGCAGGGAACAATGCGGATAAATTTCACAAATAC 5380AATGTTGAGCAAAAGAAAGACACAAAAGAATACAC ACATACACACCATATGGGCTAGG TEE-371AATGGAATCGAACGGAATCATCATCAAACGGAACCG 5381AATGGAATCATTGAATGGAATCAAAGGCAATCATGG TCGAATG TEE-372AATGGAATGGAATGTACAAGAAAGGAATGGAATGA 5382AACCGAATGGAATGGAATGGACGCAAAATGAATGG AATGGAAGTCAATGG TEE-373AACGGAAAAAAACGGAATTATCGAATGGAATCGAA 5383 GAGAATCATCGAATGGACC TEE-374GGAATAATCATTGAACGGAATCGAATGGAATCATCA 5384TCGGATGGAAACGAATGGAATCATCATCGAATGGAA ATGAAAGGAGTCATC TEE-375GGAACGAAATCGAATGGAACGGAATAGAATAGACT 5385CGAATGTAATGGATTGCTATGTAATTGATTCGAATGG AATGGAATCG TEE-376TGAAAGGAATAGACTGGAACAAAATGAAATCGAAT 5386GGTAGGAATCATACAGAACAGAAAGAAATGGAACG GAATGGAATG TEE-377AACCCGAATAGAATGGAATGGAATGGAATGGAACG 5387GAACGGAATGGAATGGAATGGATTGGAATGGAATG GAATG TEE-378AAAGAGAATCAAATGGAATTGAATCGAATGGAATCG 5388AATGGATTGGAAAGGAATAGAATGGAATGGAATGG AATGGAATGGAATGGAATG TEE-379AATGGAATCATCAGTAATGGAATGGAAAGGAATGGA 5389AAGGACTGGAATGGAATGGAATGGAATGGAATGG TEE-380GGAACAAAATGAAATCGAACGGTAGGAATCGTACA 5390GAACGGAAAGAAATGGAACGGAATGGAATGCACTC AAATGGAAAGGAGTCCAATGGAATCGAAAGGAATAGAATGGAATGG TEE-381 AGAATGAGATCAAGCAGTATAATAAAGGAAGAAGT 5391AGCAAAATTACAACAGAGCAGTGAAATGGATATGCTTTCTGGCAATAATTGTGAAAGGTCTGGTAATGAGAA AGTAGCAACAGCTAGTGGCTGC CAC TEE-382AACAAATGGAATCAACATCGAATGGAATCGAATGGA 5392AACACCATCGAATTGAAACGAATGGAATTATCATGA AATTGAAATGGATGGACTCATCATCG TEE-383TAACATGCAGCATGCACACACGAATACACAACACAC 5393AAACATGTATGCACGCACACGTGAATACACAACACACACAAACATGCATGCATGCATACATGAATACACAGC ACACAAATATCCAGCAT TEE-384GAATGGAATCAACATCAAACGGAAAAAAAACGGAA 5394TTATCGAATGGAATCGAATAGAATCATCGAATGGACC TEE-385AATCGAATGAAATGGAGTCAAAAGGAATGGAATCG 5395AATGGCAAGAAATCGAATGTAATGGAATCGCAAGGA ATTGATGTGAACGGAACGGAATGGAAT TEE-386AATGGAATTGAACGGAAACATCAGCGAATGGAATCG 5396AAAGGAATCATCATGGAATAGATTCGAATGGAATGG AAAGGAATGGAATGGAATG TEE-387ATGGAATCAACATCAAACAGAATCAAACGGAATTAT 5397CGAATGGAATCGAAGACAATCATCGAATGGACTCGAATGGAATCATCTAATGGAATGGAATGGAAGAATCCA TGGTCTCGAATGCAATCATCATCG TEE-388GAATAATCATTGAACGGAATCGAATGGAATCATCTT 5398CGGATGGAAACGAATGGAATCATCATCGAATGGAAA TGAAAGGAGTCATC TEE-389AATGGACTCGAATGGAATAATCATTGAACGGAATCG 5399AATGGAATCATCATCGGATGGAAATGAGTGGAATCA TCATCGAATGGAATCG TEE-390AAATGAAATCGAACGGTAGGAATCGTACAGAACGG 5400AAAGAAATGGAACGGAATGGAATGCAATCGAATGG AAAGGAGTCCAATGGAAGGGAATCGAAT TEE-391TACCAAACATTTAAAGAACAAATATCAATCCTACGC 5401AAACCATTCTGAAACACAGAGATGGAGGATATACAG CGAAACTCATTCTACATGGCC TEE-392TATTGGAATGGAATGGAATGGAGTCGAATGGAACGG 5402AATGCACTCGAATGGAAGGCAATGCAATGGAATGCA CTCAACAGGAATAGAATGGAATGGAATGGAATGGTEE-393 GGAATTTAATAGAATGTACCCGAATGGAACGGAATG 5403GAATGGAATTGTATGGCATGGAATGGAA TEE-394GCAATCCAATAGAATGGAATCGAATGGCATGGAATA 5404TAAAGAAATGGAATCGAAGAGAATGGAGACAAATG GAATGGAATTGAATGGAATGGAATTG TEE-395AATGGAATCGAATGGAATCATCATCAAATGGAATCT 5405AATGGAATCATTGAACGGAATTAAATGGAATCGTCATCGAATGAATTCAATGCAATCAACGAATGGTCTCGA ATGGAACCAC TEE-396AATTGCAAAAGAAACACACATATACACATATAAAAC 5406TCAAGAAAGACAAAACTAACCTATGGTGATAGAAAT CAGAAAAGTACAGTACATTGGTTGTCTTGGTGGGTEE-397 TGACATCATTATTATCAAGAAACATTCTTACCACTGT 5407TACCAACTTCCCAACACAGACTATGGAGAGAGAGAT AAGACAGAATAGCATT TEE-398AAAGAATTGAATTGAATAGAATCACCAATGAATTGA 5408ATCGAATGGAATCGTCATCGAATGGAATCGAAGGGA ATCATTGGATGGGCTCA TEE-399ATCATCGAATGGAATCGAATGGAATCAATATCAAAC 5409GGAAAAAAACGGAATTATCGAATGGAATCGAATAG AATCATCGAATGGACC TEE-400GAATGAAATCGTATAGAATCATCGAATGCAACTGAA 5410TGGAATCATTAAATGGACTTGAAAGGAATTATTATG GAATGGAATTG TEE-401TAAGCAACTTCAGCAAAGTCTCAGGATACAAAATCA 5411ATGTGCAAAAATCTCAAGCATTCTTATACACGAACA ACAGACAAACAGAGAGCT TEE-402ACTCAAAAGGAATTGATTCGAATGGAATAGAATGGC 5412AAGGAATAGTATTGAATTGAATGGAATGGAATGGAC CCAAATG TEE-403GAATGGAATTTAAAGGAATAGAATGGAAGGAATCG 5413GATGGAATGGAATGGAATAGAATGGAGTCGAATGG AATAGAATCGAATGGAATGGCATTG TEE-404TGAGAAAATGATGGAAAAGAGGAATAAAACGAAAC 5414AAAACCACAGGAACACAGGTGCATGTGAATGTGCAC AGACAAAGATACAGGGCGGACTGGGAAGGAAGTTTCTGCACCAGAATTTGGGG TEE-405 AACAAAAAATGAGTCAAGCCTTAAATAAAATCAGAG 5415CCAAAAAAGAAGACATTACATCTGATAAGACAAAAA TTCAAAGGACCATC TEE-406AACCCAGTGGAATTGAATTGAATGGAATTGAATGGA 5416ATGGAAAGAATCAATCCGAGTCGAATGGAATGGTAT GGAATGGAATGGCATGGAATCAAC TEE-407ATCAACATCAAACGGAAAAAAAACGGAATTATCGAA 5417 TGGAATCGAAGAGAATCATCGAATGGACCTEE-408 AAGGAATGGAATGGTACGGAATAGAATGGAATGGA 5418AC GAATTGTAATGGAATGGAATTTAATGGAAC GGAA TGGAATGGAATGGAATCAACG TEE-409AACGGAATGGAAAGCAATTTAATCAAATGCAATACA 5419 GTGGAATTGAAGGGAATGGAATGGAATGGCTEE-410 AATCGAATGGAACGGAATAGAATAGACTCGAATGTA 5420ATGGATTGCTATGTAATTGATTCGAATGGAATGGAATCGAATGGAATGCAATCCAATGGAATGGAATGCAAT GCAATGGAATGGAATCGAACGGAATGCAGTGGAAGGGAATGG TEE-411 TAGCAACATTTTAGTAACATGATAGAAACAAAACAG 5421CAACATAGCAATGCAATAGTAACACAACAGCAACAT CATAACATGGCAGCA TEE-412AATGGAATCGAAGAGAATGGAAACAAATGGAATGG 5422AATTGAATGGAATGGAATTGAATGGAATGGGAAGGA ATGGAGTG TEE-413AGCAAACAAGTGAATAAACAAGCAAACAAGTGAAC 5423AAGCAAACAAGTGAATAAACAAGCAAACAAGTGAA CAAGCAAACAAGTGAATAAACAAGCAAACAAGT GAACAAGGAAACAAGTGAATAAACAAAGGCTCT TEE-414AATGGAATCAACACGAGTGCAATTGAATGGAATCGA 5424ATGGAATGGAATGGAATGGAATGAATTCAACCCGAA TGGAATGGAAAGGAATGGAATC TEE-415GAATCGAATGGAATCAACATCAAACGGAAAAAAAC 5425GGAATTATCGAATGGAATCGAAGAGAATCATCGAAT GGACC TEE-416AACACGAATGTAATGCAATCCAATAGAATGGAATCG 5426AATGGCATGGAATATAAAGAAATGGAATCGAAGAG AATGGAAACAAACGGAATGGAATTGAATGGAATGGAATTGAATGGAATGGGAACGAATGGAGTGAAATTG TEE-417GAATGGAACGGAATAGAACAGACTCGAATGTAATGG 5427ATTGCTATGTAATTGATTCGAATGGAATGGAATCGAATGGAATGCAATCCAATGGAATGGAATGCAATGCAATGGAATGGAATCGAATGGAATGCAGTGGAAGGGAAT GG TEE-418GAATCGAATGGAATCAATATCAAACGGAAAAAAAC 5428GGAATTATCGAATGGAATCGAAGAGAATCATCGAAT GGACC TEE-419ATAAACATCAAACGGAATCAAACGGAATTATCGAAT 5429GGAATCGAAGAGAATAATCGAATGGACTCAAATGGAGTCATCTAATGGAATGGTATGGAAGAATCCATGGAC TCCAACGCAATCATCAGCGAATGGAATCTEE-420 AAAAGAAAAGACAAAAGACACCAATTGCCAATACT 5430GAAATGAAAAAACAGGTAATAACTATTGATCCCATG GACATTAAAATGATGTTGAAGGAACACCACTEE-421 AATGTCAAGTGGAATCGAGTGGAATCATCGAAAGAA 5431ATCGAATGGAATCGAAGGGAATCATTGGATGGGCTC AAAT TEE-422ATCATCGAATGGAATAGAATGGTATCAACATCAAAC 5432GGAGAAAAACGGAATTATCGAATGGAATCGAAGAG AATCTTCGAACGGACC TEE-423GAATGGAATCATCGCATAGAATCGGATGGAATTATC 5433ATCGAATGGAATCGAATGGTATCAACATCAAACGGAAAAAAACGGAATTATCGAATGGAATCGAATTGAATC ATCGAACGGACCCG TEE-424AATGGACTCGAATGGAATAATCATTGAACGGAATCG 5434AATGGAATCATCATCGGATGGAAATGAATGGAATAATCCATGGACTCGAATGCAATCATCATCGAATGGAAT CGAATGGAATCATCGAATGGACTCG TEE-425AATGCAATCATCAACTGGCTTCGAATGGAATCATCA 5435AGAATGGAATCGAATGGAATCATCGAATGGACTC TEE-426AAGAGACCAATAAGGAATAAGTAAGCAACAAGAGG 5436AAGGAGAAAAGGGCAAGAGAGATGACCAGAGTT TEE-427TGGAATCATCATAAAATGGAATCGAATGGAATCAAC 5437ATCAAATGGAATCAAATGGAATCATTGAACGGAATT GAATGGAATCGTCAT TEE-428GGAATCATCGCATAGAATCGAATGGAATTATCATCG 5438AATGGAATCGAATGGAATCAACATCAAACGAAAAA AAACCGGAATTATCGAATGGAATCGAAGAGAATCATCGAACGGACC TEE-429 AAATCATCATCGAATGGGATCGAATGGTATCCTTGA 5439ATGGAATCGAATGGAATCATCATCAGATGGAAATGA ATGGAATCGTCAT TEE-430GGAATGTAATAGAACGGAAAGCAATGGAATGGAAC 5440GCACTGGATTCGAGTGCAATGGAATCTATTGGAATG GAATCGAATGGAATGGTTTGGCATGGAATGGACTEE-431 AAACAATGGAAGATAATGGAAAGATATCGAATGGA 5441ATAGAATGGAATGGAATGGACTCAAATGGAATGGAC TTTAATGGAATGG TEE-432GGAACGAAATCGAATGGAACGGAATAGAATAGACT 5442CGAATGTAATGGATTGCTATGTAATTGATTCGAATGGAATGGAATCGAATGGAATGCAATCCAATGGAATGGAATGCAATGCAATGAATGGAATGGAATGGAATGGAAT GGAA TEE-433AAACCGAATGGAATGGAATGGACGCAAAATGAATG 5443GAATGGAAGTCAATGGACTCGAAATGAATGGAATGG AATGGAATGGAATG TEE-434GGAATCGAATGGAATCAACATCAAACGGAAAAAAA 5444CAGAATTATCGTATGGAATCGAATAGAATCATCGAA TGGACC TEE-435CAACCCGAGTGGAATAAAATGGAATGGAATGGAATG 5445AAATGGAATGGATCGGAATGGAATCCAATGGAATCA ACTGGAATGGAATGGAATGGAATG TEE-436TATCATCGAATGGAATCGAATGGAATCAACATCAAA 5446CGGAAAAAAACGGAATTATCGAATGGAATCGAAGA GAATCATCGAATGGACC TEE-437CGGAATAATCATTGAACGGAATCGAATGGAATCATC 5447ATCGGATGGAAACGAATGGAATCATCATCGAATGGA AATGAAAGGAGTCATC TEE-438CAACACACAGAGATTAAAACAAACAAACAAACAAT 5448CCAGCCCTGACATTTATGAGTTTACAGACTGGTGGA GAGGCAGAGAAG TEE-439CACTACAAACCACGCTCAAGGCAATAAAAGAACACA 5449AACAAATGGAAAAACATTCCATGCTCATGGATGGG TEE-440AATCGAATGGAATTAACATCAAACGGAAAAAAACG 5450GAATTATCGAATGGAATCGAAGAGAATCATCGAATG GACC TEE-441TGGAAAAGAATCAAATTGAATGGCATCGAACGGAAT 5451GGGATGGAATGGAATAGACCCAGATGTAATGGACTC GAATGGAATG TEE-442GACTAATATTCAGAATATACAAGGAACTCAAACAAC 5452TCAACAGTAGAAAAAAAAACCTGAATAGACATTTCT CAAAAGAAGACATACAAATGGCC TEE-443GGTCCATTCGATGATTCTCTTCGATTCCATTCGATAA 5453TTCCGTTTTTTCCCGTTTGATGTTGATTCC TEE-444GGAACGAAATCGAATGGAACGGAATAGAATAGACT 5454CGAATGTAATGGATTGCTATGTAATTGATTCGAATGGAATGGAATCGAATGGAATGCAATCCAATGGAATGGAATGCAATGCAATGAATGGAATGGAATGGAATGGAAT GGA TEE-445AGCAACTTCAGTAAAGTGTCAGGATACAAAATCAAT 5455GTGCAAAAATCACAAGCATTCTTATACATCAATAAC AGACAAACAGAGAGCCAAA TEE-446GAATAATCATTGAACGGAATCGAATGGAATCATCAT 5456CGGATGGAAACGAATGGAATCATCATCGAATGGAAA TGAAAGGAGTCATC TEE-447TAATCATCTTCGAATTGAAAACAAAGCAATCATTAA 5457 ATGTACTCTAACGGAATCATCGAATGGACCTEE-448 GGAATCGAATGGAATCAACATCAAACGGAAAAAAA 5458CGGAATTATCGAATGGAATCGAAGAGAATCATCGAA TGGACC TEE-449AGAGAAAAGATGATCATGTAACCATTGAAAAGACAA 5459TGTACAAAACTAATACTAATCACACAGGACCAGAAA GCAATTTAGAC CAT TEE-450AATGGAATCGAATGGAATCAACATCAAACGGAAAA 5460AACGGAATTATCGAATGGAATCAAAGAGAATCATCG AATGGACC TEE-451AATGGAATTATCATCGAATGGAATCGAATGGAATCA 5461ACATCAAACGGAAAAAAACGGAATTATCGAATGGA ATCGAAGAGAATCATCGAATGGACC TEE-452GTCAACACAGGACCAACATAGGACCAACACAGGGTC 5462AACACAGGACCAACATAGGACCAACACAGGGTCAA CACAAGACCAACATGGGACCAACACAGGGTCAACATAGGACCAACATGGGACCAACACAGGGTCAACACAG GACCAAC TEE-453GAATCAACTCGATTGCAATCGAATGGAATGGAATGG 5463TATTAACAGAATAGAATGGAATGGAATGGAATGGAA CGGAACG TEE-454ACTCGAATGCAATCAACATCAAACGGAATCAAACGG 5464AATTATCGAATGGAATCGAAGAGAATCATCGAACGG ACTCGAATGGAATCATCTAATGGAATGGAATGGTEE-455 AATGGAATGGAATAATCGACGGACCCGAATGCAATC 5465ATCATCGTACAGAATCGAATGGAATCATCGAATGGA CTGGAATGGAATGG TEE-456AATACAAACCACTGCTCAACGAAATAAAAGAGGATA 5466CAAACAAATGGAAGAACATTCTATGCTCATGGGTAG GATGAATTCATATCGTGAAAATGGCCATACTGCCTEE-457 AAACACGCAAACACACACACAAGCACACTACCACAC 5467AAGCGGACACACATGCAAACACGCGAACACACACA CATATACACACAAGCACATTACAAAACACAAGCAAACACCAGCAGACACACAAACACACAAACATACATGG TEE-458AATCGAACGGAATCAACATCAAACGGAAAAAAAAC 5468GGAATTATCGAATGGAATCGAAGAGAATCATCGAAT GGACC TEE-459TAATTGATTCGAATGGAATGGAATAGAATGGAATTG 5469AATGGAATGGACCATAATGGATTGGACTTTAATAGA AAGGGCATG TEE-460AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAAT 5470GTACAAAAGTCACAAGCATTCTTATACACCAACAAA AGACAAACAGAGAGCC TEE-461ACATCAAACGGAAAAAAAAAACAAAACGGAATTAT 5471CGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-462GAAATTCCAATTAAAATGAAATCGACTTATCTTAAC 5472AAATATAGCAATGCTGACAACACTTCTCCGGATATG GGTACTGCT TEE-463ACATCTCACTTTTAGTAATGAACAGATCATTCAGACA 5473GAAAATTAGCAAAGAAACATCAGAGTTAAACTACAC TCTAAACCAAATGGACCTA TEE-464GAAGAAAGCATTCATTCAAGACATCTAACTCGTTGA 5474TATAATGCATACAGTTCAAAATGATTACACTATCATT ACATCTAGGGCTTTC TEE-465ACACACACATTCAAAGCAGCAATATTTACAACAGCC 5475 AAAAGGTGGAAACAATTGAGCAATTGTEE-466 ATCATCGAATAGAATCGAATGGTATCAACACCAAAC 5476GGAAAAAAACGGAATTATCGAATGGAATCGAAGAG AATCTTCGAACGGACC TEE-467ATCAACATCAAACGGAAAAAACGGAATTATCGAATG 5477 GAATCGAAGAGAATCATCGAACGGACCTEE-468 AATCGAAAGGAATGTCATCGAATGGAATGGACTCAA 5478ATGGAATAGAATCGGATGGAATGGCATCGAATGGAA TGGAATGGAATTGGATGGAC TEE-469AACATGAACAGTGGAACAATCAGTGAACCAATACAA 5479GGGTTAAATAAGCTAGCAATTAAAAGCTGTATCACTGGTCTAAAGATAGAAGATCAAGTAGAAAATCAGCGC AAGAGGAAAGATATACGAAAACTAATGGCCTEE-470 CGAATGGAATCATTATGGAATGGAATGAAATGGAAT 5480AATCAAATGGAATTGAATGGAATCATCGAATGGAATCGAACAAAATCCTCTTTGAATGGAATAAGATGGAAT CACCAAATGGAATTG TEE-471AAGGGAATTGAATAGAATGAATCCGAATGGAATGGA 5481ATGGAATGGAATGGAATGGAATGGAATGGAATGGA ATGGAATG TEE-472GAATGGAATCGAATCAAATTAAATCAAATGGAATGC 5482AATAGAAGGGAATACAATGGAATAGAATGGAATGG AATGGAATGGACT TEE-473AAACGGAATCAAACGGAATTATCGAATGGAATCGAA 5483GAGAATCATCGAACGGACTCGAATGGAATCATCTAA TGGAATGGAATGGAAGAATCCATGGACTTEE-474 ATGGAATCAACATCAAACGGAAAAAAAAACGGAAT 5484TATCGAATGGAATCGAAGAGAATCATCGAATGGACC AGAATGGAATCATCTAATGGAATGGAATGGTEE-475 AATGGAATCATCATCGAATGGAATCGAATGGAATCA 5485TGGAATGGAATCAAATGGAATCAAATGGAATCGAAT GGAATGGAATGGAATG TEE-476AACGGAATCAAACGGAATTACCGAATGGAATCGAAT 5486AGAATCATCGAACGGACTCGAATGGAATCATCTAAT GGAATGGAATGGAAG TEE-477AAACGGAATCAAACGGAATTATCGAATGGAATCGAA 5487AAGAATCATCGAACGGACTCGAATGGAATCATCTAA TGGAATGGAATGGAAGAATCCATGG TEE-478GAATGATACGGAATACAATGGAATGGAACGAAATG 5488AAATGGAATGGAATGGAATGGAATGGAATGGAATGG TEE-479ACAGCAAGAGAGAAATAAAACGACAAGAAAACTAC 5489AAAATGCCTATCAATAGTTACTTTAAATATCAGTGGA CCAAATCAGTGAAACAAAAGACACAGAGTGGCTEE-480 AATGGACTCGAATGGATTAATCATTGAACGGAATCG 5490AATGGAATCATCATCGGATGGTAATGAATGGAATCA TCATCGAATGGAATCGG TEE-481GAATGGAATCGAAAGGAATGTCATCGAATGGAATGG 5491AATGGAACGGAATGGAATC GAATGGAATGGACTC GA ATGGAATAGAATCGAATGCAATGGCATCGTEE-482 ATCGAATGGAATCAACATCAGACGGAAAAAAACGG 5492AATTATCAAATGGAATCGAAGAGAATCATCGAATGG ACC TEE-483AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAAT 5493GTGCAAAAATCAAAAGCATTCTTATGCACCAATAAC AGACACAGAGCCAAAT TEE-484AATGGAATGGAACGCAATTGAATGGAATGGAATGGA 5494ACGGAATCAACCTGAGTCAAATGGAATGGAATGGAA TGGAATG TEE-485GGAACGAAATCGAATGGAACGGAATAGAATAGACT 5495CGAATGTCATGGATTGCTATGTAATTGATTGGAATGG AATGGAATCG TEE-486TAGCAGGAAACAGCAAACTCAAATTAAGTAATTTCA 5496AGAGCGTATCATCAATGAACTATTTTCAAAGATGTG GGCAAGAT TEE-487GAATTGAAAGGAATGTATTGGAATAAAATGGAATCG 5497AATAGGTTGAAATACCATAGGTTCGAATTGAATGGA ATGGGAGGGACACCAATGGAATTG TEE-488AAGCAACTTCAGCAAAGTCTCGGGATACAAAATCAA 5498TGTGCAAAAATCACAAGCATTCTTATACACCACTAA CAGACAAATGGAGAGTC TEE-489GAATGGAATCAACATCAAACGGAAAAAAACGGAAT 5499TATCGAATGGAATCGAAGAGAATCATCGAATGGACCAGAATGGAATCATCTAATGGAATGGAATGGAATAAT CCATGG TEE-490AAAAGCAATTGGACTGATTTTAAATATACGTGGCAA 5500CAAGGATAAACTGCTAATGATGGGTTTGCAAATACA GATCG TEE-491AATGGAATCAACATCGAACGGAAAAAAACGGAATT 5501ATCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-492AAACGGAATTATCAAATGGAATCGAAGAGAATCATC 5502GAACGGACTCGAATGGAATCATCTAATGGAATGGAA TGGAAG TEE-493TGCAAGATAACACATTTTAGTTGACACCATTGAAAA 5503CAGTTTTAACCAAGAATATTAGAACCAATGAAGCAGAGAAATCAAAAGGGTGGATGGAACTGCCAAAGGATG TEE-494TAGAACAGAATTGAATGGAATGGCATCAAATGGAAT 5504GGAAACGAAAGGAATGGAATTGAATGGACTCAAAT GTTATGGAATCAAAGGGAATGGACTC TEE-495AAGAGAATCATCGAATGGAATCGAATGGAATCAACA 5505TCAAACGGAAAAAAACGGAATTATCGAATGGAATCG AAGAGAATCATCGAATGGACC TEE-496ATCAACATCAAACGGAAAAAAACGGAATTATCGAAT 5506 GGAATCGAAGAGAATCATCGAATGGACCTEE-497 GAATCAACATCAAACGGAAAAAAACCGAATTATCGA 5507ATGGAATCGAAGAGAATCATCGAATGGACC TEE-498ATCAACATCAAACGGAATCAAACGGAATTATCGAAT 5508GGAATCGAAGAGAATCATCAAATGGACTCGAATGGA ATCATCTAATGGAATGGAATGGAAGAATCCATGGTEE-499 ATCGAATGGAATCATTGAATGGAAAGGAATGGAATC 5509ATCATGGAATGGAAACGAATGGAATCACTGAATGGA CTCGAATGGGATCATCA TEE-500ATTCAGCCTTTAAAAAAAGAAGACAGTCCTGTCATTT 5510GTGACAATATGAATGAAACAGACATCACATTAAATG AAATGAGCCAGGCGCAG TEE-501GAATGAAATGAAATCAAATGGAATGTACATGAATGG 5511AATAGAAAAGAATGCATCTTTCTCGAACGGAAGTGCATTGAATGGAAAGGAATCTACTGGAATGGATTCGAA TGGAATGGAATGGGATGGAATGGTATGGTEE-502 AACATCAAACGGAATCAAACGGAATTATCGAATGGA 5512ATCGAAGAGAATCATCGAACGGACTCGAATGGAATCATCTAATGGAATGGAATGGAAGAATCCATGGACTCGAATGCAATCATCATCGAATGAAATCGAATGGAATCA TCGAATGGACTCG TEE-503ATGGAATTCAATGGAATGGACATGAATGGAATGGAC 5513TTCAATGGAATGGTATCAAATGGAATGGAATTCAGT TEE-504AATGGAAAGGAATCGAATGGAAGGGAATGAAATTG 5514AATCAACAGGAATGGAAGGGAATAGAATAGACGGC AATGGAATGGACTCG TEE-505AGCAACTTCAGCAAAGTATCAGGATACAAAATCAAT 5515GTACAAAAATCCCAAGCATTCTTATACACCAACAAC AGACAAACAGAGAGCC TEE-506AGCAACTTCAGCAAAGTCTCAGGATACAAAATCGAT 5516GTGCAAAAATCACAAGCATTCTTATACACCAACAAC AGATAAACAGAGAG CC TEE-507AACGGAAAAAAAACGGAATTATCGAATGGAATCGA 5517AGAGAATCATCGAATGGACCAGAATGGAATCATCTA ATGGAATGGAATGGAATAATCCATGGACTCGAATGTEE-508 GGAATCAAACGGAATTATCGAATGGAATCGAAGAGA 5518ATCATAGAACGGACTCAAATGGAATCATCTAATGGA ATGGAATGGGAGAATCCATGGACTCGAATGTEE-509 AATGGAATCAATATCAAACGGAAAAAAACGGAATTA 5519TCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-510AACGGAATCAAACGGAATTATCGAATGGAATCGAAA 5520AGAATCATCGAACGGACTCGAATGGAATCATCTAAT GGAATGGAATGGAAGAATCCATGG TEE-511AAACGGAATTATCGAATGGAATCAAAGAGAATCATC 5521GAATGGCCACGAATGGAATCATATAATGGAATGGAA TGGAATAATCCATGGACC TEE-512AATGGAATCGAATGGATTGATATCAAATGGAATGGA 5522ATGGAAGGGAATGGAATGGAATGGAATTGAACCAA ATGTAATGGATTTG TEE-513TAAAAGACGGAACAGATAGAAAGCAGAAAGGAAAG 5523GTGAATTGCATTACCACTATTCATACTGCCACACACA TGACATTAGGCCAAGTC TEE-514AATGGAATCGAATGGAACAATCAAATGGACTCCAAT 5524GGAGTCATCTAATGGAATCGAGTGGAATCATCGAAT GGACTCG TEE-515TAACACATAAACAAACACAGAGACAAAATCTCCGAG 5525ATGTTAATCTGCTCCAGCAATACAGAACAATTTCTAT TACCAACAGAATGCTTAATTTTTCTGCCTTEE-516 GGAATCGAATGGAATCAACATCAAACGGAAAAAAA 5526CGGAATTATCGAATGGAATCAAAGAGAATCATCGAA TGGACC TEE-517AGAATGGAAAGGAATCGAAACGAAAGGAATGGAGA 5527 CAGATGGAATGGAATG TEE-518GAATCATCATAAAATGGAATCGAATGGAATCAACAT 5528CAAATGGAATCAAATGGTCTCGAATGGAATCATCTT CAAATGGAATGGAATGG TEE-519AACAACAATGACAAACAAACAACAACGACAAAGAC 5529ATTTATTTGGTTCACAAATCTCCAGGGTGTACAAGAAGCATGGTGCCAGCATCTGCTCAGCTTCTGATGAGGG CTCTGGGAAGCTTTTACTC TEE-520AACGGACTCGAACGGAATATAATGGAATGGAATGGA 5530TTCGAAAGGAATGGAATGGAATGGACAGGAAAAGA ATTGAATGGGATTGGAATGGAATCG TEE-521AACATCAAACGAAATCAAACGGAATTATCAAATTGA 5531ATCGAAGAGAATCATCGAATTGCCACGAATGCAATCATCTAATGGTATGGAATGGAATAATCCATGGACCCA GATG TEE-522AGAAATTAACAGCAAAAGAAGGATGCAGTGCAACTC 5532AGGACAACACATACAATTCAAGCAACAAATGTATAG TGGCTGGGCACCAAGGATACAG TEE-523GCAATAAAATCGACTCAGATAGAGAAGAATGCAATG 5533GAATGGAATGGAATGGAATGGAATGGGATGGAATG GTATGGAATGG TEE-524AATGGACTCGAATGAAATCATCATCAAACGGAATCG 5534AATGGAATCATTGAATGGAAAGGATGGGATCATCAT GGAATGGAAACGAATGGAATCACTG TEE-525CCACATAAAACAAAACTACAAGACAATGATAAAGTT 5535CACAACATTAACACAATCAGTAATGGAAAAGCCTAG TCAATGGCAG TEE-526TGGAATGGAATGGAATGGAATCAAATCGCATGGTAA 5536TGAATCAAATGGAATCAAATCGAATGGAAATAATGGAATCGAAGGGAAACGAATGGAATCGAATTGCACTGATTCTACTGACTTCGAGGAAAATGAAATGAAATGCGG TGAAGTGGAATGG TEE-527GAATGTTATGAAATCAACTCGAACGGAATGCAATAG 5537AATGGAATGGAATGGAATGGAATGGAATGGAATGG TEE-528AATGGAATCATTGAATGGAATGGAATGGAATCATCA 5538AAGAAAGGAATCGAAGGGAATCATCGAATGGAATC AAACGGAATCATCGAATGGAATGGAATGGAATGTEE-529 GGAATCAACATCAAACGGAAAAAAAACGGAATTATC 5539GAATGGAATCGAAGAGAATCATCGAATGGACC TEE-530GGAATAATCATCATCAAACAGAACCAAATGGAATCA 5540TTGAATGGAATCAAAGGCAATCATGGTCGAATG TEE-531GCATAGAATCGAATGGAATTATCATTGAATGGAATC 5541GAATGGAATCAACATCAAACGGAAAAAAACGGAATTATCGAATGGAATCGAAGAGAATCATCGAATGGACCC TEE-532AATGGAATCGAAGAGAATCATCGAACGGACTCGAAT 5542GGAATCATCTAATGGAATGGAATGGAATAATCCATG GACCCGAATG TEE-533AAATGAATCGAATGGAATTGAATGGAATCAAATAGA 5543ACAAATGGAATCGAAATGAATCAAATGGAATCGAAT CGAATGGAATTGAATGGCATGGAATTG TEE-534AGTTAATCCGAATAGAATGGAATGGAATGCAATGGA 5544ACGGAATGGAACGGAATGGAATGGAATGGAATGGA ATGGAATG TEE-535ATCACAATCACACAACACATTGCACATGCATAACAT 5545GCACTCACAATACACACACAACACATACACAACACACATGCAATACAACACAAAACGCAACACAACATATAC ACAACACACAGCACACACATGCC TEE-536AAAGACTTAAACGTTAGACCTAAAACCATAAAAACC 5546CTAGAGGAAAACCTAGGCATTACCATTCAGGACTTA GGCATGGGCAAGGAC TEE-537AAAGTCCAAAGATGAACAAAATATCCAGAAGGAAA 5547ACAAATGCACTTGGGGAGTGGGAAAGAAAACCAAG ACTGAGCAATGCGTCAAGCTCAGACGTGCCTCACTACG TEE-538 AAACGGAATCAAACGGAATTATCGAATGGAGTCGAA 5548AAGAATCATCGAACGGACTCGAATGGAATCATCTAA TGGAATGGAATGGAAGAATCCATGG TEE-539AATTGATTCGAAATTAATGGAATTGAATGGAATGCA 5549ATCAAATGGAATGGAATGTAATGCAATGGAATGTAA TAGAATGGAAAGCAATGGAATG TEE-540TACAGAACACATGACTCAACAACAGCAGAAAGCATA 5550TTCTTTTCAAATGCACATGAAACATTATCATGATGGA CCAAAT TEE-541GGAACAAAATGAAATCGAACGGTAGGAATCATACA 5551 GAACAGAAAGAAATGGAACGGAATGGAATGTEE-542 AACGGAAAAAACGGAATTATCGAATGGAATCGAAG 5552AGAATCATCGAATGGAATCGAATGGAGTCATCG TEE-543AATCGAACGGAATCAACATCAAACGGAAAAAAACG 5553GAATTATCGAATGGAATCGAAGAGAATCATCGAATG GACC TEE-544AGAATGGAATGCAATAGAATGGAATGCAATGGAATG 5554GAGTCATCCGTAATGGAATGGAAAGGAATGCAATGG AATGGAATGGAATGG TEE-545ATGGAATCAACATCAAACGGAATCAAACGGAATTAT 5555CGAATGGAATCGAAGAGAATCATCGAACGGATTCGAATGGAATCATCTAATGGAATGGAATGGAAGAATCCATGGACTCGAATGCAATCATCAGCGAATGGAATCGAA TGGAATCATCGAATGGACTCG TEE-546GGAATAAAACGGACTCAATAGTAATGGATTGCAATG 5556TAATTGATTCGATTTCGAATGGAATCGCATGGAATGT AATGGAATGGAATGGAATGGAAGGC TEE-547AATGGAATCAACATCAAACGGAAAAAAACGGAATT 5557ATCGTATGGAATCGAAAAGAATTATCGAATGGACC TEE-548TCAAACGGAAAAAAACGGAATTATCGAATGGAATCG 5558 AAGAGAATCATCGAATGGACC TEE-549ACATCAAACGGAATCAAACGGAATTATCGAATGGAA 5559TCGAAAAGAATCATCGAACGGACTCGAATGGAATCATCTAATGGAATGGAATGGAAGAATCCATGGACTCGA ATG TEE-550TGGAATCGAATGGAATCAACATCAAACGGAAAAAA 5560ACGGAATTATCGAATGGAATCGAAGAGAATCATCGA ATGGACC TEE-551AATGGAATCGAATGCAATCATCGAACGGAATCGAAT 5561GGCATCACCGAATGGAATGGAATGGAATGGAATGGA ATGG TEE-552AGAATTGATTGAATCCAAGTGGAATTGAATGGAATG 5562GAATGGATTAGAAAGGAATGGAATGGATTGGAATGG ATTGGAATGGAAAGG TEE-553AACTGCATCAACTAACAGGCAAAATAACCAGCTAAT 5563ATCATAATGACAGGATTAAATTCACAAATGACAATA TTAACCGTAAATGTAAATGGGCTA TEE-554GTAAACAAACAATCAAGCAAGTAAGAACAGAAATA 5564ACAGCATTTGGCTTTTGAGTTAATGACAAGAACACTCGGCATGGGAGCCTGGGTGAGCAAATCACAGATCTTC TEE-555AAAGGAATGGACTGGAACAAAATGAAATCGAACGG 5565TAGGAATCGTACAGAACGGACAGAAATGGAACGGC ATGGAATGCACTCG TEE-556GAATCAACCCGAGCGGAAAGGAATGGAATGGAATG 5566GAATCAACACGAATGGAATGGAACGGAATGGAATG GGATGGGATGAAATGGAATGG TEE-557AAGAAATGGAATCGAAGAGAATGGAAACAAACGGA 5567ATGGAATTGAATGGAATGGAATTGAATGGAATGGGA TEE-558GACATGCAAACACAACACACAGCACACATGGAACAT 5568GCATCAGACATGCAAACACAACACACATACCACACA TGGCATATGCATCAGACGTGCCTCACTACTEE-559 AAAGGAATGCACTCGAATGGAATGGACTTGAATGGA 5569ATGTCTCCGAATGGAACAGACTCGTATGAAATGGAATCGAATGGAATGGAATCAAATGGAATTGATTTGAGT GAAATGGAATCAAATGGAATGGCAACG TEE-560GGAACAAAATGAAATCGAACGGTAGGAATCGTACA 5570GAACGGAAAGAAATGGAACGGAATGGAATGCACTC GAATGGAAAGGAGTCCAAT TEE-561AAATTGATTGAAATCATCATAAAATGGAATCGAAGG 5571GAATCAACATCAAATGGAATCAAATGGAATCATTGA AC GGAATTGAATGGAATC GTCAT TEE-562AGAATGGAAAGCAATAGAATGGAACGCACTGGATTC 5572GAGTGCAATGGAATCAATTGGAATGGAATCGAATGG AATGGATTGGCA TEE-563AACACCAAACGGAAAAAAACGGAATTATCGAATGG 5573AATCGAAGAGAATCTTCGAAC GGAC CC GAATGGGAT CATCTAATGGAATGGAATGGAATAATCCATGGTEE-564 AATGGAGACTAATGTAATAGAATCAAATGGAATGGC 5574ATCGAATGGAATGGACTGGAATGGAATGTGCATGAA TGGAATGGAATCGAATGGATTG TEE-565AAATCGAATGGAACGCAATAGAATAGACTCGAATGT 5575AATGGATTGCTATGTAATTGATTCGAATGGAATGGAATCGACTGGAATGCAATCCAATGGAATGGAATGCAATGCAATGGAATGGAATCGAACGGAATGCAGTGGAAG GGAATGG TEE-566AATCAACAAGGAACTGAAACAAGTAAACAAGAAAA 5576CAAATAACACCATAAAACATGGGCAAAGGACATAAACAGACATTTTTCAAAAAAGACATACAAATGGCCGAG TEE-567AATGGAATCAACATCAAACGGAAAAAAACGGAATT 5577ATCGAATGGAATCGAAGAGAATCATCGAATGGACCC AGGCTGGTCTTGAACTCC TEE-568ATTGAATGGGCTAGAATGGAATCATCTTTGAACGGA 5578ATCAAAGGGAATCATCATC GAATGGAATCGAATG GA AATGTCAACG TEE-569AATGGACTCGAATGGAATCAACATCAAATGGAATCA 5579AGCGGAATTATCGAATGAAATCGAAGAGAATCATCGAATGGACTCGAAAGGAATCATCTAATGGAATGGAAT GGAATAATCCATGGACTCGAATGCAATCATCATCGTEE-570 AAAC GGAAAAAAAC GGAATTATTGAATGGAATCGA 5580AGAGAATCTTCGAACGGACCCGAATGGAATCATCTA ATGGAATGGAATGGAATAATCCATGG TEE-571ACTCGAGTGGAATTGACTGTAACAAAATGGAAAGTA 5581ACGGATTGGAATCGAATGGAACGGAATGGAATGGA ATGGACAT TEE-572TACAAACTTTAAAAAATGATCAACAGATACACAGTT 5582AGCAAGAAAGAATTGAGGGCAAAGAATATGCCAGA CAAACTCAAGAGGAAGATGATGGTAGAGATAGGTCACATTGGAGTGTCA TEE-573 AAATCAACAACAAACGGAAAAAAAAGGAATTATCG 5583AATGGAATCAAAGAGAATCATCGAATGGACC TEE-574AACGGAATCAAACGGAATTATCGAATGGAATCGAAA 5584AGAATCATCGAACGGACTCGAATGGAATCATCTAAT GTAATGGAATGGAAGAATCCATGGACTCGAATGTEE-575 AACGGAAAAAAACGGAATTATCGAATGGAATCGAA 5585GAGAATCATCGAATGGACCAGAATGGAATCATCTAA TGGAATGGAATGGAATAATCCATGGACTCGAATGTEE-576 CAACATCAAACGGAAAAAAACGGAATTATGGAATG 5586GAATC GAAGAGAATCATCGAATGGAC CC GAATGGAA TCATCTGAAATATAATAGACTCGAAAGGAATGTEE-577 ATGGAATCGAATGGAATGGACTGGAATGGAATGGAT 5587TCGAATGGAATCGAATGGAACAATATGGAATGGTAC CAAATG TEE-578GAATGGAATCAACATCAAACGGAAAAAAACGGAAT 5588TATCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-579AAATGGACTCGAATGGAATCATCATAGAATGGAATC 5589GAATGCAATGGAATGGAATCTTCCGGAATGGAATGG AATGGAATGGAATGGAG TEE-580GAATCATCATAAAATGGAATCGAATGGAATCAACAT 5590CAAATGGAATCAAATGGAATCATTGAACGGAATTGA ATGGAATCGTCAT TEE-581ATCGAATGGAATCAACATCAAACGGAAAAAAACGG 5591AATTATCGAATGGAATCGAAGAGAATCATCGAATGG ACC TEE-582AGCAACTTCAGCAAAGTCTCAGGATACAAAATCAAT 5592GTACAAAAATCACAAGCATTCTTATACACCAATAAC AGACAAACAGAGAGCCAAAA TEE-583AGAAACAGAAAACAGTCAAACCAATGGGCAATCCAT 5593ATCAGATGCAGTATTATGAACAGAAGTGTAAAGAAT GCACCAGGCACAATGGC TEE-584GATTGGAACGAAATCGAATGGAACGGAATAGAATA 5594GACTC GAATGTAAT GGATTGCTAT GTAATTGATTC GAATGGAATGGAATCGAATGGAATGCAATCCAATGGAA TGGAATGCAATGCAATGGAATGG TEE-585ATGGAATGGAATAATCAACGTACTCGAATGCAATCA 5595TCATCGTATAGAATCGAATGGAATCATCGAATGGACTCGAATGGAATAATCATTGAACGGAGTCGAATGGAA TCATCATCGGATGGAAAC TEE-586AAAGAAATCGAATGGAATCAGTGTCGAATGGAATGG 5596AATGGAATCGAAGAATTGAATTGAGTAGAATCGAAG GGAATCATTGGATGGGCTCAAAT TEE-587AGAAAAGATAACTCGATTAACAAATGAACAAACACC 5597TGAATACACAAGTCTCAAAAGAAGACATAAAAATGG CCAAC TEE-588ATGGAATCAACATCAAACGGAATCACACGGAATTAT 5598CGAATGGAATCGAAAAGAATCATCGAACGGACTC GA ATGGAATCATCTAATGGAATGGAATGGAAGTEE-589 AATGGAATCAACATCAAACGGAATCAAGCGAAATTA 5599TCGAATGGAATCGAAGAGAATCATCGAATGGACTCG AATGGAATCATCTAATGGAATGGAATGGGATTEE-590 AAACACAGTACAAATACTAATTCAAATCAAACTTAC 5600TCAAAGTCATAATCAAACATGCCAGACGGGCTGAGG GGCAGCATTA TEE-591GGAATCGAGTGGAATCATCGAAAGAAATCGAATGGA 5601ATCATTGTCGAATGGAATGGAATGGAATCAAAGAAT GGAATCGAAGGGAATCATTGGATGGGCTTEE-592 AAAGAAAGACAGAGAACAAACGTAATTCAAGAT GA 5602CTGTTTACATATCCAAGAACATTAGATGGTCAAAGACTTTAAGAAGGAATACATTCAAAGGCAAAAAGTCAC TTACTGATTTTGGTGGAGTTTGCCACATGGACTEE-593 GAAAGGAATCATCATTGAATGCAATCACATGGAATC 5603ATCACAGAATGGAATCGTACGGAATCATCATCGAATGGAATTGAATGGAATCATCAATTGGACTCGAATGGAATCATCAAATGGAATCGATTGGAAGTGTCAAATGGA CTCG TEE-594CAATCAGAGCGGACACAAACAAATTGCATGGGAAG 5604 AATCAATATCGTGAAAATGGCC TEE-595CAGCGCACCACAGCACACACAGTATACACATGACCC 5605ACAATACACACAACACACAACACATTCACACACCAC TEE-596GCAAACAGAATTCAACACTACATTAGAACGATCATT 5606CATCACGACCTAGTAGGATGTTTTTCCTGGGATGCAA GGATGGTTCAACAT TEE-597CAATCAAAACAGCAATGAGATACCATTTTACACCAA 5607TCAAAATGGCTACTAAAAAGTCAAAAGCAAATGCC TEE-598TGGAATAGAATGGAATCAATGTTAAGTGGAATCGAG 5608TGGAATCATCGAAAGAAATCGAATGGAATCATTGTC GAATGGTATGGAATGGAATCA TEE-599AATGGAATGGAATCATCGCATAGAATGGAATGGAAT 5609TATCATCGAATTGAATCGAATGGTATCAACATCAAA CGGAAAAAAACGGAAATATCGAATGGAATCGAAGAGAATCATCGAACGGACC TEE-600 GAAAAACAAAACAAAACAAACAAACAAACAATCAA 5610AAAAGTGGTAGCAGAAACCAGAAAGTCCATGTATAT AGCTAATTGGCCTGGTTGT TEE-601AGACCTTTCTCAGAAGACACACAAATTGCCAACAGG 5611TATATGAAAAAATGTTCAATATCACTAATCATCAGG GCGATGCC TEE-602CATGGAATCGAATGGAATTATCATCGAATGGAATCG 5612AATGGTACCAACACCAAACGGAAAAAAACGGAATT ATCGAATGGAATCGAAGAGAATCTTCGAACGGACCTEE-603 AGAGCAGAAACAAATGGAATTGAAATGAAGACAAC 5613AATCAAAAGCATCAATGAAATGAAAAGTTGGGTTTT GGAAGAGAGAAACAAT TEE-604ACACAAACACACACACACACACACACACACACACAC 5614ACACACACACACACACACACACACACACACATAC TEE-605AACAAACAAATGAGATGATTTCAGATAGTGATAAAC 5615ACTATAACATAATTAATTCGTGCCAATCAGAGCATA ACAGTGGTGTGGTGGCTGTGGAACAGATAGCAGACTEE-606 AATGGAATCGAGTGGAATGGAAGGCAATGGAATAG 5616AATGGAATGGAATCGAAAGGAACGGAATGGAATGG AATGGAATG TEE-607AGAAATGGAATCGGAGAGAATGGAAACAAATGGAA 5617TGGAATTGAATGGAATGGAATTGAATGGAATGGGAA CG TEE-608AAGAGAACTGCAAAACACTGCTCAAAGAAATCAGA 5618GATGACAAAAACACATGGAAAAACGTTTCATGCTCA TGGATTGGAAGACTTA TEE-609AATCAACACGAATAGAATGGAACGGAATGGAATGG 5619AATGGAATGGAATGGAATGGAGTGGAATGGAACAG AATGGAGTGGAAT TEE-610AACATCAAACGAAATCAAACGGAATTATCAAATTGA 5620ATCGAAGAGAATCATCGAATTGCCACGAATGCAACCATCTAATGGTATGGAATGGAATAATCCATGGACCCA GATG TEE-611CGGAATTATCATCGAATGTAATCGAATGGAATCAAC 5621ATCAAACGGAAAAAAACGGAATTATCGAATGGAATC GAAGAGAATCATCGAATGGACC TEE-612TGGACACACACGAACACACACCTACACACACGTGGA 5622CACACACGGACACATGGACACACACGAACACATGGA CACACACACGGGGACACACACAGACACACACAGAGACACACACGGACACATGG TEE-613 ATCAAACGGAATCAAACGGAATTATCGAATGGAATC 5623GAAGAGAATCATCGAATGGACTCGAATGGAATCATC TAATGGAATGGAATGGAAGAATCCATGGTEE-614 AAATGGAATGGAATGCACTTGAAAGGAATAGACTGG 5624AACAAAATGAAATCGAACGGTAGGAATCATACAGA ACAGAAAGAAATGGAACGGAATGGAATG TEE-615AC CACACACAAAATACAC CACACACCACACACACAC 5625CACACACTATACACACACCACACACCACACAC TEE-616AAAGAAATAGAAGGGAGTTGAACAGAATCGAATGG 5626AATCGAATCAAATGGAATCGAATGGCATCAAATGGA ATCGAATGGAATGTGGTGAAGTGGATTGGTEE-617 GGAATCATCATAAAATGGAATCGAATGGAATCATCA 5627TCAAATGGAATCAAATGGAATCATTGAACGGAATTG AATGGAATCGTCAT TEE-618AAAGATCAATGTACAAAAATCAGCAGCATTTCTATA 5628AACCAACAATGTCCAGGCTGAGAGAGAAATCAAGA AAACAATTC TEE-619TGGAATGGAATGGAATGAAATAAACACGAATAGAAT 5629GGAACGGAATGGAACGGAATGGAATGGAATGGAAT GGAAAG TEE-620TAATCAGCACAATCAACTGTAGTCACAAAACAAATA 5630GTAACGCAATGATAAAGAAACAGAGAACTAGTTCAA ATAAACATGATAAGATGGGG TEE-621AAGCGGAATTATCAAATGGAATCGAAGAGAATGGA 5631AACAAATGGAATGGAATTGAATGGAATGGAATTGAA TGGAATG TEE-622AATGGAATCAACATCAAACGGAAAAAAACGGAATT 5632ATCGAATGGAATCGAAGAGAATCATCGAATGGACC TEE-623ACTTGAATCGAATGGAAAGGAATTTAATGAACTTAA 5633ATCGAATGGAATATAATGGTATGGAATGGACTCATG GAATGGAATGGAAAGGAATC TEE-624TGGAATCATCATCGAAAGCAAGCGAATGGAATCATC 5634AAATGGAAACGAATGGAATCATCGAATGGACTCGGA TGGAATTGTTGAATGGACT TEE-625TGGAATCAACATCAAACGGAAAAAAACGGAATTATC 5635GAATGGAATCGAAGAGAATCATCGAATGGACC TEE-626TAAGTGAATTGAATAGAATCAATCTGAATGTAATGA 5636AATGGAATGGAACGGAATGGAATGGAATGGAATGG AATGGAATGGAATGG TEE-627AGGAAAATTTAATCAGCAGGAATAGAAACACACTTG 5637AGAAATCCATGTGGAATGAAAAGAGAATGGCTGAGC AGCAACAGATTGTCAAAAAGGAAATC TEE-628AACATCAAACGGAAAAAAAACGGAATTATCGAATG 5638 GAATCGAAGAGAATCATCGAATGGACCTEE-629 TAATTGAGAATAAGCATTCCAGTGGAAAAAAAACTA 5639AACAATTTGTTGTAAAACATCCTTAAAAGCATCAGAAAGTTAATACAGCAATGAAGAATTACAGGACCAAAT TAAGAATGGTATGGAAGCCTGTTA TEE-630TATCATCGAATGGAATCGAATGGAATCAACATCAAA 5640CGGAAAAAAACGGAATTATCGAATTGAATCGAAGAG AATCATCGAATGGACC TEE-631AGCAAAACAAACACAATCTGTCGTTCATGGTACTAC 5641GACATACTGGGAGAGATATTCAAATGATCACACAAA ACAACATG TEE-632AAGGATTCGAATGGAATGAAAAAGAATTGAATGGA 5642ATAGAACAGAATGGAATCAAATCGAATGAAATGGA GTGGAATAGAAAGGAATGGAATG TEE-633AACGGAATCAAACGGAATTATCGAATGGAATCGAAG 5643AGAATCATCGAACGGACTCGAATGGAATCATCTAATGGAATGGAATGGAAGAATCCATGGACTCGAATGCAATCATCATCGAATGGAATCGAACGGAATCATCGAATG GCC TEE-634AATCAACTAGATGTCAATGGAATGCAATGGAATAGA 5644ATGGAATGGAATTAACACGAATAGAATGGAATGGAA TGGAATGGAATGG TEE-635AATGGACTCGAATGGAATAATCATTGAACGGAATCG 5645AATGGAATCATCATCGGATGGAAATGAATGGAATCA TCATCGCATGGAATCG TEE-636GAATGGAATGATACGGAATAGAATGGAATGGAACG 5646AAATGGAATTGAAAGGAAAGGAATGGAATGGAATG GAATGG TEE-637AATCATCATCGAATGGAATCGAATGGTATCATTGAG 5647TGGAATCGAATGGAATCATCATCAGATGGAAATGAA TGGAATCGTCAT TEE-638GAATCAAATCAATGGAATCAAATCAAATGGAATGGA 5648 ATGGAATTGTATGGAATGGAATGGCATGGTEE-639 TAATGCAGTCCAATAGAATGGAATCGAATGGCATGG 5649AATATAAAGAAATGGAATCGAAGAGAATGGGAACA AATGGAATGGAATTGAGTGGAATGGAATTGAATGGAATGGGAACGAATGGAGTG TEE-640 AACATCAAACGGAAAAAAACGGAATTATCGAATGG 5650AATCGAAGAGAATCATCGAATGGACC TEE-641 ATCAAAAGGAACGGAATGGAATGGAATGGAATGGA5651 ATGGAATGGAATGGAATGGAATGAAATCAACCCGAA TGGAATGGATTGGCATAGAGTGGAATGGTEE-642 GCCAACAATCATATGAGAAAAAGCTCAACATCACTG 5652ATCATTTCAGGAATGCAAATCAAAACCACAATGAGA TACTATCA TEE-643AATCAAATGGAATGAAATCGAATGGAATTGAATCGA 5653ATGGAATGCAATAGAATGTCTTCAAATGGAATCGAA TGGAAATTGGTGAAGTGGACGGGAGTG TEE-644TAATGGAATCAACATCAAACGGAAAAAAACGGAATT 5654ATCGAATGCAATCGAAGAGAATCATCGAATGGACC TEE-645AGCAACTTCAGCAAAGTCTCAGCATACAAAATCAAT 5655GTGCAAAAATCACACGCATTCCTATACACCAATAAC AGACAAACAGAGAGCC TEE-646GAATCAAATGGAATGGACTGTAATGGAATGGATTCG 5656AATGGAATCGAATGGAGTGGACTCAAATGGAATG TEE-647AACAAGTGGACGAAGGATATGAACAGACACTTCTCA 5657AGACATTTATGCAGCCAACAGACACACGAAAAAATG CTCATCATCACTGGCCATCAG TEE-648AAACGGAAAAAAACGGAATTATCGAATGGAATCGA 5658 ATAGAATCATCGAATGGACC TEE-649TGGAACCGAACAAAGTCATCACCGAATGGAATTGAA 5659ATGAATCATAATCGAATGGAATCAAATGGCATCTTC GAATTGACTCGAATGCAATCATCCACTGGGCTTTEE-650 AACGGAATCACGCGGAATTATCGAATGGAATCGAAG 5660AGAATCATCGAATGGACTCGAATGGAATCATCTAAT GGAATGGAATGG TEE-651GGAATCAACTCGATTGCAATGGAATGCAATGGAAAG 5661GAATGGAATGCAATTAAAGCGAATAGAATGGAATGG AATGGAATGGAACGGAATGGAATG TEE-652AAAACAAACAACAACGACAAATCATGAGACCAGAG 5662TTAAGAAACAATGAGACCAGGCTGGGTGTGGTG TEE-653AATCGAAAGGAATGCAATATTATTGAACAGAATCGA 5663AAAGAATGGAATCAAATGGAATGGAACAGAGTGGA ATGGACTGC TEE-654AAGGAATCGAATGGAAGTGAATGAAATTGAATCAAC 5664AGGAATGGAAGGGAATAGAATAGACTGTAATGGAA TGGACTCG TEE-655AACCCGAGTGCAATAGAATGGAATCGAATGGAATGG 5665 AATGGAATGGAATGGAATGGAATGGAGTCTEE-656 GAATGGAATTGAAAGGAATGGAATGCAATGGAATG 5666GAATGGGATGGAATGGAATGCAATGGAATCAACTCG ATTGCAATG TEE-657GAAAAAAACGGAATTATCGAATTGAATCAAATAGAA 5667TCATCGAACGGACCAAAATGGAATCATCTAATGGAA TGGAATGGAATAATCCATGGACTCTAATGTEE-658 TGGAATCATCTAATGGAATGGAATGGAATAATCCAT 5668GGACTCGAATGCAATCATCATAAAATGGAATCGAATGGAATCAACATCAAATGGAATCAAATGGGATCATTG AAC GGAATTGAATG GAATCGT CAT TEE-659GAAAAAAACGGAATTATCGAATTGAATCGAATAGAA 5669TCATCGAACGGACCAGAATGGAATCATCTAATGGAA TGGAATGGAATAATCCATGGACTCGAATGTEE-660 AACCACTGCTTAAGGAAATAAGAGAGAACACAAAC 5670AAATGGAAAAACGTTCCATGCTCATGGATAGGAGAA TCAATATCGTGAAAATGGCC TEE-661TATCGAATGGAATGGAAAGGAGTGGAGTAGACTCGA 5671ATAGAATGGACTGGAATGAAATAGATTCGAATGGAA TGGAATGGAATGAAGTGGACTCG TEE-662GTATCAACATCAAACGGAAAAAAACGGAATTATCGA 5672ATGGAATCATCTAATGGAATGGAATGGAATAATCCA TGGACTCGAATG TEE-663TAAATGGAGACATCATTGAATACAATTGAATGGAAT 5673CATCACATGGAATCGAATGGAATCATCGTAAATGCA ATCAAGTGGAATCAT TEE-664GAATGGAATTGAAAGGTATCAACACCAAACGGAAA 5674AAAAAACGGAATTATCGAATGGAATCGAAGAGAATC ATCGAACGGACC TEE-665AGCAATTTCAGCAAAGTCTCAGGATACAAAATCAAT 5675GTACAAATTCACAAGCATTCTTATGGACCAACAACAG TEE-666GGAATCGAATGGCATCAACATCAAACGGAAAAAAA 5676CGGAATTATCGAATGGAATCGAATGGAATCATC TEE-667AAACAAAACACAGAAATGCAAAGACAAAACATAAA 5677ACGCAGCCATAAAGGACATATTTTAGATAACTGGGG AAATTTGTATGGGCTGTGT TEE-668AATGGAATCAACATCAAACGGAATCAAACGGAATTA 5678TCGAATGGAATCGAAGAGAATCATCGAACGGACTCG AATGGAATCATCTAATGGAATGGAATGGAAGTEE-669 AATCGAATGGAATCAGCATCAAACGGAAAAAAACG 5679GAATTATCGAATGGAATCGAAGAGAATCATCGAATG GACC TEE-670AAACGGAATTATAGAATGGACTGGAAGAGAATCATC 5680GAACGGACTAGAATGGAATCATCTAATCGAATGGAA TGGAACAATCCATGGTCTAGCA TEE-671TGAACAGAGAATTGGACAAAACGCACAAAGTAAAG 5681AAAAAGAATGAAGCAACAAAAGCAGAGATTTATTG AAAACAAAAGTACACACCACACAGGGTGGGAGTGGTEE-672 ATCATAACGACAAGAACAAATTCACACACAACAATA 5682TTAACTTCAAATCCAAATGGGTTAAATGCTCCAATTA AAGGATGCAGACGGGCAAATTGGATA TEE-673ATCATAACGACAAGAACAAATTCACACACAACAATA 5683TTCACTTCAAATCCAAATGGGTTAAATGCTCCAATTA AAGGATGCAGACGGGCAAATTGGATA TEE-674GAATGGAATCGAATGGATTGATATCAACTGGAATGG 5684AATGGAAGGGAATGGAATGGAATGGAATTGAACCA AATGTAATGACTTGAATGGAATG TEE-675GAATCAACATCAAACGGAAAAAAACGGAATTATCGA 5685 ATGGAATCGAAGAGAATCATCGAATGGACCTEE-676 GGAATCAACATCAAACGGAAAAAAACGGAATTATCG 5686AATGGAATCGAAGAGAATCATCGAATGGACC TEE-677ATGGAATCAACATCAAACGGAATCAAACGGAATTAT 5687CGAATGGAATCAAAGAGAATCATCGAACGGACTC GAATGGAATCATCTAATGGAATGGAATGGAAGAATCCA TGGACTCGAATGCAATCATCATCGAAT TEE-678GGAATGGAATGGAATGGAGCCGAATGGAATGGAAT 5688 GTACTCAAATGGAATGC TEE-679AAAACACCTAGGAATACAGATAACAAGGGACATTAA 5689CTACCTCTTAAAGAGAACTACAAACCACTGCTCAAG GAAATGAGAGAGGACACAAACACATGGAAAAACATTCCATCCTCATGGATAGGAAGAATCAATATTGTGAA AATGGCC TEE-680AACACGACTTTGAGAAGAGTAAGTGATTGTTAATTA 5690AAGCAAGAGAATTATTGATGTATCACAGTCATGAGA AATATTGGAAGGAATATGGTCCATAC TEE-681ACACATATCAAACAAACAAAAGCAATTGACTATCTA 5691 GAAATGTCTGGGAAATGGCAAGATATTACATEE-682 GGAATCATCATATAATGGAATCGAATGGAATCAACA 5692TCAAATGGAATCAAATGGAATCATTGAACGGAATTG AATGGAATCGTCAT TEE-683AATGGAATCAACATCAAACGGAATCAAATGGAATTA 5693TCGAATGGAATCGAAGAGAATCATCGAATTGTCACGAATGGAATCATCTAATGGAATGGAATGGAATAATCCATGGCCCCTATGCAATGGACTCGAATGAAATCATCATCAAACAGAATCGAATGGAATCATCTAATGGAATGG AATGGCATAATCCATGGACTCGAATG TEE-684TAAAATGAAACAAATATACAACACGAAGGTTATCAC 5694CAGAAATATGCCAAAACTTAAATATGAGAATAAGAC AGTCTCAGGGGCCACAGAG TEE-685AAAATACAGCGTTATGAAAAGAATGAACACACACAC 5695 ACACACACACACACAGAAAATGTTEE-686 CAAACAAATAGGTACCAAACAAATAACAACATAAAC 5696CTGACAACACACTTATTTACAAGAGACATCCCTTATATGAAAGGGTACAGAAAAGTCGATGGTAAGATGATGG GGAAAGGTATACCAACCACTAGCAGAAGGTEE-687 TGGAATCGAATGGAATCAATATCAAACGGAAAAAAA 5697CGGAATTATCGAATGGAATCGAAAAGAATCATCGAA TGGGCCCGAATGGAATCATCT TEE-688ACAAATGGAATCAACAACGAATGGAATCGAATGGA 5698AACGCCATCGAAAGGAAACGAATGGAATTATCATGA AATTGAAATGGATG TEE-689AATCAATAAATGTAAACCAGCATATAAACAGAACCA 5699ACGACAAAAACCACATGATTATCTCAATAGATGCAG AAAAGGCC TEE-690AAAATAAACGCAAATTAAAATCACAAGATACCAACA 5700CATTCCCACGGCTAAGTACGAAGAACAAGGGCGAAT GGTCAGAATTAAGCTCAAACCT TEE-691CAACATCAAACGGAATCAAACGGAATTATCGAATGG 5701AATCGAAGAGAATCATCGAATGGACTCGAATGGAAT CATCTAATGGAATGGAATGGAAG TEE-692ACATCAAACGGAAAAAAACGGAATTATCGAATGGA 5702 ATCGAAGAGAATCATCGAATGGACCTEE-693 AATGGACTCGAATAGAATTGACTGGAATGGAATGGA 5703CTCGAATGGAATGGAATGGAATGGAAGGGACTCG TEE-694AAGAAAGACAGAGAACAAACGTAATTCAAGATGAC 5704TGATTACATATCCAAGAACATTAGATGGTCAAAGACTTTAAGAAGGAATACATTCAAAGGCAAAACGTCACT TACTGATTTTGGTGGAGTTTGCCACATGGACTEE-695 GAATGGAATCGAATGGAATGAACATCAAACGGAAA 5705AAAACGGAATTATCGAATGGAATCAAAGAGAATCAT CGAATGGACCCG TEE-696ATGGACTCGAATGTAATAATCATTGAACGGAATCGA 5706ATGGAATCATCATCGGATGGAAACGAATGGAATCAT CATCGAATGGAATCGAATGGGATC TEE-697GAAATGGAATGGAAAGGAATAAAATCAAGTGAAAT 5707 TGGATGGAATGGATTGGAATGGATTGGAATGTEE-698 AAACGGAAAAAAAACGGAATTATCGAATGGAATCG 5708AAGAGAATCATCGAACGAACCAGAATGGAATCATCT AATGGAATGGAATGGAATAATCCATGG TEE-699ATTAACCCGAATAGAATGGAATGGAATGGAATGGAA 5709CGGAACGGAATGGAATGGAATGGAATGGAATGGAA TGGATCG TEE-700AACATCAAACGGAAAAAAACGGAATTATCGTATGGA 5710 ATCGAAGAGAATCATCGAATGGACCTEE-701 GAATAGAATTGAATCATCATTGAATGGAATCGAGTA 5711GAATCATTGAAATCGAATGGAATCATCATCGAATGG AATTGGGTGGAATC TEE-702CACCGAATAGAATCGAATGGAACAATCATCGAATGG 5712ACTCAAATGGAATTATCCTCAAATGGAATCGAATGG AATTATCG TEE-703AATGCAATCGAATAGAATCATCGAATAGACTCGAAT 5713GGAATCATCGAATGGAATGGAATGGAACAGTC TEE-704AAATCATCATCGAATGGAATCGAATGGTATCATTGA 5714ATGGAATCGAATGGAATCATCATCAGATGGAAATGA ATGGAATCGTCAT TEE-705GAATGGAATCGAAAGGAATAGAATGGAATGGATCGT 5715TATGGAAAGACATCGAATGGAATGGAATTGACTCGA ATGGAATGGACTGGAATGGAACG

Example 35 In Vitro Expression of Modified Nucleic Acids with miR-122

MicroRNA controls gene expression through the translational suppressionand/or degradation of target messenger RNA. The expression of G-CSF mRNAand Factor IX mRNA with human or mouse alpha-globin 3′ untranslatedregions (UTRs) were down regulated in human primary hepatocytes usingmiR-122 sequences in the 3′UTR.

Primary human hepatocytes were seeded at a density of 350000 per well in500 ul cell culture medium (InVitro GRO medium from Celsis, Chicago,Ill.).

G-CSF mRNA having a human alpha-globin 3′UTR (G-CSF Hs3′UTR; mRNAsequence shown in SEQ ID NO: 5716; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1) or a mouse alpha-globin3′UTR (G-CSF Mm3′UTR; mRNA sequence shown in SEQ ID NO: 5717; polyA tailof approximately 140 nucleotides not shown in sequence; 5′ cap, Cap1)were fully modified with 5-methylcytidine and 1-methylpseudouridine.G-CSF mRNA containing a human 3′UTR having a miR-122 sequence in the3′UTR (G-CSF Hs3′UTR miR-122; mRNA sequence shown in SEQ ID NO: 5018;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1), or a miR-122 seed sequence in the 3′UTR (G-CSF Hs3′UTRmiR-122 seed; mRNA sequence shown in SEQ ID NO: 5020; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) or amiR-122 sequence without the seed sequence in the 3′UTR (G-CSF Hs3′UTRmiR-122 seedless; mRNA sequence shown in SEQ ID NO: 5022; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) werefully modified with 5-methylcytidine and 1-methylpseudouridine. G-CSFmRNA containing a mouse 3′UTR having a miR-122 sequence in the 3′UTR(G-CSF Mm3′UTR miR-122; mRNA sequence shown in SEQ ID NO: 5024; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1), or a miR-122 seed sequence in the 3′UTR (G-CSF Mm3′UTR miR-122seed; mRNA sequence shown in SEQ ID NO: 5026; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) or amiR-122 sequence without the seed sequence in the 3′UTR (G-CSF Mm3′UTRmiR-122 seedless; mRNA sequence shown in SEQ ID NO: 5028; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) werefully modified with 5-methylcytidine and 1-methylpseudouridine.

Factor IX mRNA having a human alpha-globin 3′UTR (Factor IX Hs3′UTR;mRNA sequence shown in SEQ ID NO: 5718; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1) or a mouse alpha-globin3′UTR (Factor IX Mm3′UTR; mRNA sequence shown in SEQ ID NO: 5719; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) were fully modified with 5-methylcytidine and1-methylpseudouridine. Factor IX mRNA containing a human 3′UTR having amiR-122 sequence in the 3′UTR (Factor IX Hs3′UTR miR-122; mRNA sequenceshown in SEQ ID NO: 5030; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1), or a miR-122 seed sequence in the3′UTR (Factor IX Hs3′UTR miR-122 seed; mRNA sequence shown in SEQ ID NO:5032; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1) or a miR-122 sequence without the seed sequence in the3′UTR (Factor IX Hs3′UTR miR-122 seedless; mRNA sequence shown in SEQ IDNO: 5034; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) were fully modified with 5-methylcytidine and1-methylpseudouridine. Factor IX mRNA containing a mouse 3′UTR having amiR-122 sequence in the 3′UTR (Factor IX Mm3′UTR miR-122; mRNA sequenceshown in SEQ ID NO: 5036; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1), or a miR-122 seed sequence in the3′UTR (Factor IX Mm3′UTR miR-122 seed; mRNA sequence shown in SEQ ID NO:5038; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1) or a miR-122 sequence without the seed sequence in the3′UTR (Factor IX Mm3′UTR miR-122 seedless; mRNA sequence shown in SEQ IDNO: 5040; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) were fully modified with 5-methylcytidine and1-methylpseudouridine.

Each G-CSF or Factor IX mRNA sequence was tested at a concentration of500 ng per well in 24 well plates. 24, 48 and 72 hours aftertransfection, the expression of protein was measured by ELISA. Theprotein levels for G-CSF are shown in Table 33 and the protein levelsfor Factor IX are shown in Table 34.

TABLE 33 G-CSF Protein Expression Protein Expression (ng/ml) Description24 Hours 48 Hours 72 Hours G-CSF Hs3′UTR 43.9 18.8 5.7 G-CSF Hs3′UTRmiR-122 6.9 0.7 0.12 G-CSF Hs3′UTR miR-122 seed 48.5 25.6 8.2 G-CSFHs3′UTR miR-122 seedless 31.7 11.7 3.4 G-CSF Mm3′UTR 84.9 100.4 21.3G-CSF Mm3′UTR miR-122 24.0 3.03 0.8 G-CSF Mm3′UTR miR-122 seed 115.896.4 19.2 G-CSF Mm3′UTR miR-122 seedless 113.1 92.9 18.9

TABLE 34 Factor IX Protein Expression Protein Expression (ng/ml)Description 24 Hours 48 Hours 72 Hours Factor IX Hs3′UTR 63.2 124.8 44.3Factor IX Hs3′UTR miR-122 15.9 4.4 0.4 Factor IX Hs3′UTR miR-122 seed60.2 63.0 20.1 Factor IX Hs3′UTR miR-122 seedless 53.7 75.0 24.5 FactorIX Mm3′UTR 90.8 159.6 70.5 Factor IX Mm3′UTR miR-122 11.8 5.0 1.0 FactorIX Mm3′UTR miR-122 seed 77.2 115.0 41.7 Factor IX Mm3′UTR miR-122 69.3123.8 49 seedless

Example 36 In Vitro Expression of Modified Nucleic Acid with mir-142 ormiR-146 binding sites

HeLa and RAW264 cells were seeded at a density of 17000 and 80000 perwell respectively, in 100 ul cell culture medium (DMEM+10% FBS).

G-CSF mRNA (G-CSF; mRNA sequence shown in SEQ ID NO: 4258; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) wasfully modified with 5-methylcytidine and 1-methylpseudouridine.

G-CSF mRNA having a miR-142-3p sequence in the 3′UTR (G-CSF miR-142-3p;mRNA sequence shown in SEQ ID NO: 4992; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1), or a miR-142-3p seedsequence in the 3′UTR (G-CSF miR-142-3p seed; mRNA sequence shown in SEQID NO: 4994; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) or a miR-142-3p sequence without the seedsequence in the 3′UTR (G-CSF miR-142-3p seedless; mRNA sequence shown inSEQ ID NO: 4996; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1) were fully modified with 5-methylcytidine and1-methylpseudouridine.

G-CSF mRNA having a miR-142-5p sequence in the 3′UTR (G-CSF miR-142-5p;mRNA sequence shown in SEQ ID NO: 4986; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1), or a miR-142-5p seedsequence in the 3′UTR (G-CSF miR-142-5p seed; mRNA sequence shown in SEQID NO: 4988; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) or a miR-142-5p sequence without the seedsequence in the 3′UTR (G-CSF miR-142-5p seedless; mRNA sequence shown inSEQ ID NO: 4990; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1) were fully modified with 5-methylcytidine and1-methylpseudouridine.

G-CSF mRNA having a miR-146a sequence in the 3′UTR (G-CSF miR-146a; mRNAsequence shown in SEQ ID NO: 4998; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1), or a miR-146a seedsequence in the 3′UTR (G-CSF miR-146a seed; mRNA sequence shown in SEQID NO: 5000; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) or a miR-146a sequence without the seed sequencein the 3′UTR (G-CSF miR-146a seedless; mRNA sequence shown in SEQ ID NO:5002; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1) were fully modified with 5-methylcytidine and1-methylpseudouridine.

Each G-CSF mRNA sequence was tested at a concentration of 500 ng perwell in 24 well plates for each cell type. 24 hours after transfection,the expression of protein was measured by ELISA and the protein levelsare shown in Table 35. The G-CSF sequences with a miR-142-3p sequence inthe 3′UTR down regulated G-CSF expression in RAW264 cells whereas theG-CSF sequences with a miR-142-5p or miR-146a sequence in the 3′UTR didnot down regulate G-CSF expression.

TABLE 35 G-CSF Expression HeLa Cells RAW264 Cells Protein ProteinDescription Expression (ng/ml) Expression (ng/ml) G-CSF 243.5 173.7G-CSF miR-142-3p 309.1 67.6 G-CSF miR-142-3p seed 259.8 178.1 G-CSFmiR-142-3p seedless 321.7 220.2 G-CSF miR-142-5p 291.8 223.3 G-CSFmiR-142-5p seed 261.3 233.1 G-CSF miR-142-5p seedless 330.2 255.1 G-CSFmiR-146a 272.6 125.2 G-CSF miR-146a seed 219.4 138.3 G-CSF miR-146aseedless 217.7 132.8

Example 37 Effect of Kozak Sequence on Expression of Modified NucleicAcids

HeLa cells were seeded at a density of 17000 per well in 100 ul cellculture medium (DMEM+10% FBS). G-CSF mRNA having an IRES sequence andKozak sequence (G-CSF IRES Kozak; mRNA sequence shown in SEQ ID NO:5004; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1), G-CSF mRNA having an IRES sequence but not a Kozaksequence (G-CSF IRES; mRNA sequence shown in SEQ ID NO: 5006; polyA tailof approximately 140 nucleotides not shown in sequence; 5′ cap, Cap1),G-CSF mRNA without an IRES or Kozak sequence (GCSF no Kozak; mRNAsequence shown in SEQ ID NO: 5008; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1) or a G-CSF sequencehaving a Kozak sequence (G-CSF Kozak; mRNA sequence shown in SEQ ID NO:5720; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1) were fully modified with fully modified with5-methylcytidine and 1-methylpseudouridine and tested at a concentrationof 75 ng per well in 24 well plates. 24 hours after transfection, theexpression of G-CSF was measured by ELISA, and the results are shown inTable 36.

TABLE 36 G-CSF expression Description Protein Expression (ng/ml) G-CSFIRES Kozak 2.01 G-CSF IRES 1.64 G-CSF no Kozak 795.53 G-CSF Kozak 606.28

Example 38 MALAT1 Constructs

Modified mRNA encoding G-CSF or mCherry with a human or mouse MALAT1sequence and their corresponding cDNA sequences are shown below in Table37. In Table 37, the start codon of each sequence is underlined and theMALAT1 sequences are bolded.

TABLE 37 MALAT1 Constructs SEQ ID Sequence NO: G-CSFOptimized G-CSF cDNA sequence   5721 withcontaining a T7 polymerase site,   Mouse kozak sequence, and a Mouse MALAT1 MALAT1 sequence (bold): sequence TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATA TAAGAGCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGA CATCTTGCGCAGCCG TGATAATAGGATTCGTCAGTAGGGTTGTAAAGGTTTTTCTTTTCCTGAGAAAACAACCTTTTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTCCCTAGCTTTAAAAAAAAAAAAGCAAAAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTCTA GA mRNA sequence (transcribed):5722 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAU AUAAGAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCG UGAUAAUAGGAUUCGUCAGUAGGGUUGUAAAGGUUUUUCUUUU CCUGAGAAAACAACCUUUUGUUUUCUCAGGUUUUGCUUUUUGGCCUUUCCCUAGCUUUAAAAAAAAAAAA GCAAAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC mCherry Optimized mCherry cDNA sequence  5723 withcontaining a T7 polymerase site,  Mousekozak sequence, and a Mouse MALAT1  MALAT1 sequence (bold): sequenceTAATACGACTCACTATA GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATA TAAGAGCCACCATGGTATCCAAGGGGGAGGAGGACAACATGGCGATCATCAAGGAGTTCATGCGATTCAAGGTGCACATGGAAGGTTCGGTCAACGGACACGAATTTGAAATCGAAGGAGAGGGTGAAGGAAGGCCCTATGAAGGGACACAGACCGCGAAACTCAAGGTCACGAAAGGGGGACCACTTCCTTTCGCCTGGGACATTCTTTCGCCCCAGTTTATGTACGGGTCCAAAGCATATGTGAAGCATCCCGCCGATATTCCTGACTATCTGAAACTCAGCTTTCCCGAGGGATTCAAGTGGGAGCGGGTCATGAACTTTGAGGACGGGGGTGTAGTCACCGTAACCCAAGACTCAAGCCTCCAAGACGGCGAGTTCATCTACAAGGTCAAACTGCGGGGGACTAACTTTCCGTCGGATGGGCCGGTGATGCAGAAGAAAACGATGGGATGGGAAGCGTCATCGGAGAGGATGTACCCAGAAGATGGTGCATTGAAGGGGGAGATCAAGCAGAGACTGAAGTTGAAAGATGGGGGACATTATGATGCCGAGGTGAAAACGACATACAAAGCGAAAAAGCCGGTGCAGCTTCCCGGAGCGTATAATGTGAATATCAAGTTGGATATTACTTCACACAATGAGGACTACACAATTGTCGAACAGTACGAACGCGCTGAGGGTAGACACTCGACGGGAGGCATGGACGAG TTGTACAAA TGATAATAGGATTCGTCAGTAGGGTTGTAAAGGTTTTTCTTTTCCTGAGAAAACAACCTTTTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTCCCTAGCTTTAAAAAAAAAAAAGCAAAAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTCTA GA mRNA sequence (transcribed):5724 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAU AUAAGAGCCACCAUGGUAUCCAAGGGGGAGGAGGACAACAUGGCGAUCAUCAAGGAGUUCAUGCGAUUCAAGGUGCACAUGGAAGGUUCGGUCAACGGACACGAAUUUGAAAUCGAAGGAGAGGGUGAAGGAAGGCCCUAUGAAGGGACACAGACCGCGAAACUCAAGGUCACGAAAGGGGGACCACUUCCUUUCGCCUGGGACAUUCUUUCGCCCCAGUUUAUGUACGGGUCCAAAGCAUAUGUGAAGCAUCCCGCCGAUAUUCCUGACUAUCUGAAACUCAGCUUUCCCGAGGGAUUC AAGUGGGAGCGGGUCAUGAACUUUGAGGACGGGGGUGUAGUCACCGUAACCCAAGACUCAAGCCUCCAAGACGGCGAGUUCAUCUACAAGGUCAAACUGCGGGGGACUAACUUUCCGUCGGAUGGGCCGGUGAUGCAGAAGAA AACGAUGGGAUGGGAAGCGUCAUCGGAGAGGAUGUACCCAGAAGAUGGUGCAUUGAAGGGGGAGAUCAAGC AGAGACUGAAGUUGAAAGAUGGGGGACAUUAUGAUGCCGAGGUGAAAACGACAUACAAAGCGAAAAAGCCGGUGCAGCUUCCCGGAGCGUAUAAUGUGAAUAUCAAGUUGGAUAUUACUUCACACAAUGAGGACUACACAAUUGUCGAACAGUACGAACGCGCUGAGGGUAGACACUCG ACGGGAGGCAUGGACGAGUUGUACAAAUGAUAAUAG GAUUCGUCAGUAGGGUUGUAAAGGUUUUUCUUUUCCUGAGAAAACAACCUUUUGUUUUCUCAGGUUUUG CUUUUUGGCCUUUCCCUAGCUUUAAAAAAAAAAAAGCAAAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCG GC G-CSFOptimized G-CSF cDNA sequence   5725 with containing a T7 polymerase Human site,kozak sequence,  MALAT1 and a Human MALAT1 sequence (bold):sequence TAATACGACTCACTATA GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC ATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGA CATCTTGCGCAGCCG TGATAATAGTGCTCTTCAGTAGGGTCATGAAGGTTTTTCTTTTCCTGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGCAAAAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTC TAGA mRNA sequence (transcribed):5726 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAU AUAAGAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCG UGAUAAUAGUGCUCUUCAGUAGGGUCAUGAAGGUUUUUCUUUUC CUGAGAAAACAACACGUAUUGUUUUCUCAGGUUUUGCUUUUUGGCCUUUUUCUAGCUUAAAAAAAAAAAA AGCAAAAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC mCherry Optimized mCherry cDNA sequence   5727 withcontaining a T7 polymerase  Human site, kozak sequence,  MALAT1and a Human MALAT1 sequence (bold): sequence TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATA TAAGAGCCACCATGGTATCCAAGGGGGAGGAGGACAACATGGCGATCATCAAGGAGTTCATGCGATTCAAGGTGCACATGGAAGGTTCGGTCAACGGACACGAATTTGAAATCGAAGGAGAGGGTGAAGGAAGGCCCTATGAAGGGACACAGACCGCGAAACTCAAGGTCACGAAAGGGGGACCACTTCCTTTCGCCTGGGACATTCTTTCGCCCCAGTTTATGTACGGGTCCAAAGCATATGTGAAGCATCCCGCCGATATTCCTGACTATCTGAAACTCAGCTTTCCCGAGGGATTCAAGTGGGAGCGGGTCATGAACTTTGAGGACGGGGGTGTAGTCACCGTAACCCAAGACTCAAGCCTCCAAGACGGCGAGTTCATCTACAAGGTCAAACTGCGGGGGACTAACTTTCCGTCGGATGGGCCGGTGATGCAGAAGAAAACGATGGGATGGGAAGCGTCATCGGAGAGGATGTACCCAGAAGATGGTGCATTGAAGGGGGAGATCAAGCAGAGACTGAAGTTGAAAGATGGGGGACATTATGATGCCGAGGTGAAAACGACATACAAAGCGAAAAAGCCGGTGCAGCTTCCCGGAGCGTATAATGTGAATATCAAGTTGGATATTACTTCACACAATGAGGACTACACAATTGTCGAACAGTACGAACGCGCTGAGGGTAGACACTCGACGGGAGGCATGGACGAG TTGTACAAA TGATAATAGTGCTCTTCAGTAGGGTCATGAAGGTTTTTCTTTTCCTGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTTTCTAGCTTAAAAAAAAAAAAAGCAAAAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTC TAGA mRNA sequence (transcribed):5728 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAU AUAAGAGCCACCAUGGUAUCCAAGGGGGAGGAGGACAACAUGGCGAUCAUCAAGGAGUUCAUGCGAUUCAAGGUGCACAUGGAAGGUUCGGUCAACGGACACGAAUUUGAAAUCGAAGGAGAGGGUGAAGGAAGGCCCUAUGAAGGGACACAGACCGCGAAACUCAAGGUCACGAAAGGGGGACCACUUCCUUUCGCCUGGGACAUUCUUUCGCCCCAGUUUAUGUACGGGUCCAAAGCAUAUGUGAAGCAUCCCGCCGAUAUUCCUGACUAUCUGAAACUCAGCUUUCCCGAGGGAUUC AAGUGGGAGCGGGUCAUGAACUUUGAGGACGGGGGUGUAGUCACCGUAACCCAAGACUCAAGCCUCCAAGACGGCGAGUUCAUCUACAAGGUCAAACUGCGGGGGACUAACUUUCCGUCGGAUGGGCCGGUGAUGCAGAAGAA AACGAUGGGAUGGGAAGCGUCAUCGGAGAGGAUGUACCCAGAAGAUGGUGCAUUGAAGGGGGAGAUCAAGC AGAGACUGAAGUUGAAAGAUGGGGGACAUUAUGAUGCCGAGGUGAAAACGACAUACAAAGCGAAAAAGCCGGUGCAGCUUCCCGGAGCGUAUAAUGUGAAUAUCAAGUUGGAUAUUACUUCACACAAUGAGGACUACACAAUUGUCGAACAGUACGAACGCGCUGAGGGUAGACACUCG ACGGGAGGCAUGGACGAGUUGUACAAAUGAUAAUAG UGCUCUUCAGUAGGGUCAUGAAGGUUUUUCUUUUCCUGAGAAAACAACACGUAUUGUUUUCUCAGGUUUU GCUUUUUGGCCUUUUUCUAGCUUAAAAAAAAAAAAAGCAAAAGUGGUCUUUGAAUAAAGUCUGAGUGGGC GGC

These modified mRNA sequences can include at least one chemicalmodification described herein. The G-CSF or mCherry modified mRNAsequence can be formulated, using methods described herein and/or knownin the art, prior to transfection and/or administration.

The modified mRNA sequence encoding G-CSF or mCherry can be transfectedin vitro to various cell types such as HEK293, HeLa, PBMC and BJfibroblast and those described in Table 25 using methods disclosedherein and/or are known in the art. The cells are then analyzed usingmethods disclosed herein and/or are known in the art to determine theconcentration of G-CSF or mCherry and/or the cell viability.

Example 39 Utilization of Heterologous 5′UTRs

A 5′ UTR may be provided as a flanking region to the nucleic acids,modified nucleic acids or mmRNA of the invention. 5′UTR may behomologous or heterologous to the coding region found in the nucleicacids, modified nucleic acids or mmRNA of the invention. Multiple 5′UTRs may be included in the flanking region and may be the same or ofdifferent sequences. Any portion of the flanking regions, includingnone, may be codon optimized and any may independently contain one ormore different structural or chemical modifications, before and/or aftercodon optimization.

Shown in Lengthy Table 21 in U.S. Provisional Application No.61/775,509, filed Mar. 9, 2013, entitled Heterologous UntranslatedRegions for mRNA and in Lengthy Table 21 in U.S. Provisional ApplicationNo. 61/829,372, filed May 31, 2013, entitled Heterologous UntranslatedRegions for mRNA is a listing of the start and stop site of thepolynucleotides, primary constructs or mmRNAs of the invention. Each5′UTR (5′UTR-005 to 5′UTR 68511) is identified by its start and stopsite relative to its native or wild type (homologous) transcript (ENST;the identifier used in the ENSEMBL database).

To alter one or more properties of the nucleic acids, modified nucleicacids or mmRNA of the invention, 5′UTRs which are heterologous to thecoding region of the nucleic acids, modified nucleic acids or mmRNA ofthe invention are engineered into compounds of the invention. Thenucleic acids, modified nucleic acids or mmRNA are then administered tocells, tissue or organisms and outcomes such as protein level,localization and/or half life are measured to evaluate the beneficialeffects the heterologous 5′UTR may have on the nucleic acids, modifiednucleic acids or mmRNA of the invention. Variants of the 5′ UTRs may beutilized wherein one or more nucleotides are added or removed to thetermini, including A, T, C or G. 5′UTRs may also be codon-optimized ormodified in any manner described herein.

Example 40 Further Utilization of 5′ Untranslated Regions

A 5′ UTR may be provided as a flanking region to the nucleic acids,modified nucleic acids or mmRNA of the invention. 5′UTR may behomologous or heterologous to the coding region found in the nucleicacids, modified nucleic acids or mmRNA of the invention. Multiple 5′UTRs may be included in the flanking region and may be the same or ofdifferent sequences. Any portion of the flanking regions, includingnone, may be codon optimized and any may independently contain one ormore different structural or chemical modifications, before and/or aftercodon optimization.

Shown in Table 38 is a listing of 5′-untranslated regions which may beused with the nucleic acids, modified nucleic acids or mmRNA of thepresent invention.

To alter one or more properties of the nucleic acids, modified nucleicacids or mmRNA of the invention, 5′UTRs which are heterologous to thecoding region of the nucleic acids, modified nucleic acids or mmRNA ofthe invention are engineered into compounds of the invention. Thenucleic acids, modified nucleic acids or mmRNA are then administered tocells, tissue or organisms and outcomes such as protein level,localization and/or half life are measured to evaluate the beneficialeffects the heterologous 5′UTR may have on the nucleic acids, modifiednucleic acids or mmRNA of the invention. Variants of the 5′ UTRs may beutilized wherein one or more nucleotides are added or removed to thetermini, including A, T, C or G. 5′UTRs may also be codon-optimized ormodified in any manner described herein.

TABLE 38 5′-Untranslated Regions SEQ 5′ UTR Name/ ID IdentifierDescription Sequence NO. 5UTR- UpstreamGGGAGATCAGAGAGAAAAGAAGAGTAAGAAGAAATA 5729 68512 UTR TAAGAGCCACC 5UTR-Upstream GGAATAAAAGTCTCAACACAACATATACAAAACAAAC 5730 68513 UTRGAATCTCAAGCAATCAAGCATTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCT GAAAATTTTCACCATTTACGAACGATAGCAAC5UTR- Upstream GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAA 5731 68514 UTRGCCACC 5UTR- Upstream GGGAATTAACAGAGAAAAGAAGAGTAAGAAGAAATA 5732 68515UTR TAAGAGCCACC 5UTR- Upstream GGGAAATTAGACAGAAAAGAAGAGTAAGAAGAAATA 573368516 UTR TAAGAGCCACC 5UTR- UpstreamGGGAAATAAGAGAGTAAAGAACAGTAAGAAGAAATA 5734 68517 UTR TAAGAGCCACC 5UTR-Upstream GGGAAAAAAGAGAGAAAAGAAGACTAAGAAGAAATA 5735 68518 UTR TAAGAGCCACC5UTR- Upstream GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGATATA 5736 68519 UTRTAAGAGCCACC 5UTR- Upstream GGGAAATAAGAGACAAAACAAGAGTAAGAAGAAATA 573768520 UTR TAAGAGCCACC 5UTR- UpstreamGGGAAATTAGAGAGTAAAGAACAGTAAGTAGAATTAA 5738 68521 UTR AAGAGCCACC 5UTR-Upstream GGGAAATAAGAGAGAATAGAAGAGTAAGAAGAAATA 5739 68522 UTR TAAGAGCCACC5UTR- Upstream GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAAAT 5740 68523 UTRTAAGAGCCACC 5UTR- Upstream GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATT 574168524 UTR TAAGAGCCACC

Example 41 Protein Production Using Heterologous 5′UTRs

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 37.5 ng,75 ng or 150 of G-CSF modified RNA comprising a nucleic acid sequencefor 5UTR-001 (mRNA sequence shown in SEQ ID NO: 5; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytidine and 1-methylpseudouridine), G-CSFmodified RNA comprising a nucleic acid sequence for 5UTR-68515 (mRNAsequence shown in SEQ ID NO: 5732; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine), G-CSF modified RNAcomprising a nucleic acid sequence for 5UTR-68516 (mRNA sequence shownin SEQ ID NO: 5733; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytidineand 1-methylpseudouridine), G-CSF modified RNA comprising a nucleic acidsequence for 5UTR-68521 (mRNA sequence shown in SEQ ID NO: 5738; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytidine and 1-methylpseudouridine) orG-CSF modified RNA comprising a nucleic acid sequence for 5UTR-68522(mRNA sequence shown in SEQ ID NO: 5739; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine) were diluted in 10 ul finalvolume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine2000 (LifeTechnologies, Grand Island, N.Y.) was used as transfectionreagent and 0.2 ul were diluted in 10 ul final volume of OPTI-MEM. After5 minutes of incubation at room temperature, both solutions werecombined and incubated an additional 15 minute at room temperature. Thenthe 20 ul combined solution was added to the 100 ul cell culture mediumcontaining the HeLa cells and incubated at room temperature.

After an incubation of 24 hours cells expressing G-CSF were lysed with100 ul of Passive Lysis Buffer (Promega, Madison, Wis.) according tomanufacturer instructions. G-CSF protein production was determined byELISA.

These results, shown in Table 39, demonstrate that G-CSF mRNA comprisingthe 5UTR-68515 or 5UTR-68521 produced the most protein whereas G-CSFmRNA comprising 5UTR-68522 produced the least amount of protein.

TABLE 39 G-CSF Protein Production from Heterologous 5′UTRs G-CSF Protein(ng/ml) 5′UTR 37.5 ng 75 ng 150 ng 5UTR-001 131.3 191.1 696.1 5UTR-68515245.6 394.3 850.3 5UTR-68516 188.6 397.4 719.6 5UTR-68521 191.4 449.1892.1 5UTR-68522 135.9 331.3 595.6

Example 42 Effect of the Kozak Sequence in Modified Nucleic Acids

HeLa cells were seeded at a density of 15,000 per well in 100 ul cellculture medium DMEM+FBS 10%. G-CSF mRNA having a Kozak sequence (G-CSFKozak; mRNA sequence shown in SEQ ID NO: 5004; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytidine and 1-methylpseudouridine) or G-CSF mRNAnot having a Kozak sequence (G-CSF no Kozak; mRNA sequence shown in SEQID NO: 5008; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine) and transfected in triplicate at a concentrationof 75 ng per well in 96 well plates. 24 hours, 48 hours and 72 hoursafter transfection, the supernatant was collected and expression ofG-CSF was measured by ELISA, and the results are shown in Table 40.

TABLE 40 G-CSF Expression G-CSF Kozak G-CSF No Kozak Time point ProteinExpression (ng/ml) Protein Expression (ng/ml) 24 hours 223.93 408.23 48hours 604.76 1217.29 72 hours 365.48 703.93

Example 43 Effect of the Altering the 5′UTR in Modified Nucleic Acids

mRNA encoding a polypeptide of interest and having a polyA sequence anda cap, is fully modified with at least one chemistry described hereinand/or in co-pending International Publication No WO2013052523, thecontents of which are herein incorporated by reference in its entiretyand the mRNA comprises a 5′UTR from Table 41. HeLa cells are seeded incell culture medium and are transfected with the mRNA at apre-determined concentration (ng per well) in well plates. Atpre-determined intervals (e.g., 2 hours, 4 hours, 6 hours, 8 hours, 10hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24hours, 36 hours, 48 hours, 60 hours and/or 72 hours) after transfection,the supernatant is collected and expression of protein is measured byELISA.

TABLE 41 5′ UTR SEQ 5′ UTR ID Identifier Name/Description Sequence NO.5UTR-001 Synthetic GGGAAATAAGAGAGAAAAGAAGAGTAAGAAG 5 UTRAAATATAAGAGCCACC 5UTR- Synthetic GGGAAATAAGAGAGAAAAGAAGAGTAAGAAG 574268525 UTR AAATATAAGAGCCTCC 5UTR- SyntheticGGGAAATAAGAGAGAAAAGAAGAGTAAGAAG 5743 68526 UTR AAATATATGA 5UTR-Synthetic GGGAAATAAGAGAGAAAAGAAGAGTAAGAAG 5744 68527 UTR AAATATA

Example 44 Effect of the PolyA Tail Length in Modified Nucleic Acids

A. Bioanalyzer

Modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 5745; 5′ cap, Cap1 fully modified with 5-methylcytidine and 1-methylpseudouridine(5mc/1mpU) or fully modified with 1-methylpseudouridine (1mpU)),modified Factor IX (FIX) mRNA (mRNA sequence shown in SEQ ID NO: 5746;5′ cap, Cap 1 fully modified with 5-methylcytidine and1-methylpseudouridine (5mc/1mpU) or fully modified with1-methylpseudouridine (1mpU)), modified erythropoietin (EPO) mRNA (mRNAsequence shown in SEQ ID NO: 5747; 5′ cap, Cap 1 fully modified with5-methylcytidine and 1-methylpseudouridine (5mc/1mpU) or fully modifiedwith 1-methylpseudouridine (1mpU)) or modified mCherry mRNA (mRNAsequence shown in SEQ ID NO: 5748; 5′ cap, Cap 1 fully modified with5-methylcytidine and 1-methylpseudouridine (5mc/1mpU) or fully modifiedwith 1-methylpseudouridine (1mpU)) having a polyA tail of 20, 40, 80,100, 120, 140 or 160 nucleotides in length or no polyA tail wereanalyzed by bioanalyzer (Agilent 2100 bioanalyzer). All samples,maintained integrity of the mRNA as determined by bioanalyzer.

B. BJ Fibroblasts

Human primary foreskin fibroblasts (BJ fibroblasts) are obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, cat#11095-114) supplemented with10% fetal bovine serum at 37° C., under 5% CO₂. BJ fibroblasts areseeded on a 24-well plate at a density of 100,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 5745; 5′ cap, Cap 1 fully modified with 5-methylcytidineand 1-methylpseudouridine (5mc/1mpU) or fully modified with1-methylpseudouridine (1mpU)), modified Factor IX (FIX) mRNA (mRNAsequence shown in SEQ ID NO: 5746; 5′ cap, Cap 1 fully modified with5-methylcytidine and 1-methylpseudouridine (5mc/1mpU) or fully modifiedwith 1-methylpseudouridine (1mpU)), modified erythropoietin (EPO) mRNA(mRNA sequence shown in SEQ ID NO: 5747; 5′ cap, Cap 1 fully modifiedwith 5-methylcytidine and 1-methylpseudouridine (5mc/1mpU) or fullymodified with 1-methylpseudouridine (1mpU)) or modified mCherry mRNA(mRNA sequence shown in SEQ ID NO: 5748; 5′ cap, Cap 1 fully modifiedwith 5-methylcytidine and 1-methylpseudouridine (5mc/1mpU) or fullymodified with 1-methylpseudouridine (1mpU)) having a polyA tail of 20,40, 80, 100, 120, 140 or 160 nucleotides in length or no polyA tail weretransfected using Lipofectamine 2000, following manufacturer's protocol.FIX, G-CSF and EPO were transfected in triplicate.

The supernatant was collected at 24 hours, 48 hours and 72 hours forFIX, G-CSF and EPO and at 24 hours for mCherry. The protein expressionwas analyzed by ELISA for FIX, G-CSF and EPO and fluorescence-activatedcell sorting (FACS) for mCherry. The results for G-CSF are shown inTable 42, the results for EPO are shown in Table 43, the results for FIXare shown in Table 44 and the results for mCherry are shown in Table 45.

TABLE 42 G-CSF Protein Expression Description PolyA Tail Length TimePoint Protein (ng/ml) G-CSF 5mC/1mpU 0 24 1.13 48 0.39 72 0.2 G-CSF 1mpU0 24 2 48 0.3 72 0.16 G-CSF 5mC/1mpU 20 24 41.85 48 32.75 72 13.38 G-CSF1mpU 20 24 204.43 48 138.71 72 96.36 G-CSF 5mC/1mpU 40 24 102.75 48101.96 72 48.97 G-CSF 1mpU 40 24 451.71 48 373.75 72 217.62 G-CSF5mC/1mpU 80 24 135.85 48 167.21 72 96.66 G-CSF 1mpU 80 24 534.89 48352.39 72 203.89 G-CSF 5mC/1mpU 100 24 168.31 48 195.16 72 127.8 G-CSF1mpU 100 24 561 48 406.8 72 265.64 G-CSF 5mC/1mpU 120 24 152.54 48187.06 72 100.41 G-CSF 1mpU 120 24 656.23 48 511.01 72 239.95 G-CSF5mC/1mpU 140 24 146.24 48 202.05 72 121.89 G-CSF 1mpU 140 24 724.58 48627.6 72 341.61 G-CSF 5mC/1mpU 160 24 59.83 48 101.30 72 64.69 G-CSF1mpU 160 24 814.54 48 579.65 72 274.7

TABLE 43 EPO Protein Expression Description PolyA Tail Length Time PointProtein (ng/ml) EPO 5mC/1mpU 0 24 3.12 48 0.13 72 0 EPO 1mpU 0 24 0.7748 0.07 72 0.007 EPO 5mC/1mpU 20 24 48.93 48 21.72 72 5.88 EPO 1mpU 2024 199.24 48 42.9 72 20.29 EPO 5mC/1mpU 40 24 400.66 48 165.38 72 63.36EPO 1mpU 40 24 210 48 182.56 72 54.31 EPO 5mC/1mpU 80 24 368.09 48303.05 72 117.98 EPO 1mpU 80 24 422.95 48 229.53 72 131.05 EPO 5mC/1mpU100 24 619.59 48 366.19 72 199.63 EPO 1mpU 100 24 374.88 48 240.21 72128.08 EPO 5mC/1mpU 120 24 430.64 48 354.6 72 165.72 EPO 1mpU 120 24358.02 48 193.77 72 104.89 EPO 5mC/1mpU 140 24 531 48 426.96 72 164.3EPO 1mpU 140 24 355.96 48 202.27 72 99.88 EPO 5mC/1mpU 160 24 608.66 48324.31 72 181.94 EPO 1mpU 160 24 351.01 48 197.76 72 109.64

TABLE 44 FIX Protein Expression Description PolyA Tail Length Time PointProtein (ng/ml) FIX 5mC/1mpU 0 24 0.51 48 1.14 72 0.47 FIX 1mpU 0 240.61 48 0.39 72 0.36 FIX 5mC/1mpU 20 24 0.92 48 0.46 72 0.49 FIX 1mpU 2024 5.97 48 14.99 72 7.47 FIX 5mC/1mpU 40 24 2.27 48 1.62 72 0.5 FIX 1mpU40 24 15.32 48 41.92 72 21.05 FIX 5mC/1mpU 80 24 7.12 48 10.14 72 3.66FIX 1mpU 80 24 35.32 48 74.18 72 38.47 FIX 5mC/1mpU 100 24 8.47 48 13.3372 6.73 FIX 1mpU 100 24 40.5 48 90.56 72 54.85 FIX 5mC/1mpU 120 24 10.0648 15.89 72 6.2 FIX 1mpU 120 24 47.5 48 106.55 72 59.35 FIX 5mC/1mpU 14024 11.16 48 20.13 72 8.85 FIX 1mpU 140 24 46.44 48 109.03 72 60.17 FIX5mC/1mpU 160 24 13.06 48 22.31 72 10.19 FIX 1mpU 160 24 45.35 48 99.4772 60.48

TABLE 45 mCherry Expression Expression Description PolyA Tail LengthTime Point index mCherry 5mC/1mpU 0 24 445946.66 mCherry 1mpU 0 24509423.33 mCherry 5mC/1mpU 20 24 510846.66 mCherry 1mpU 20 24 1688910mCherry 5mC/1mpU 40 24 1443583.33 mCherry 1mpU 40 24 3398540 mCherry5mC/1mpU 80 24 1949826.66 mCherry 1mpU 80 24 5783383.33 mCherry 5mC/1mpU100 24 4963426.66 mCherry 1mpU 100 24 4639580 mCherry 5mC/1mpU 120 245372706.66 mCherry 1mpU 120 24 9184466.66 mCherry 5mC/1mpU 140 245127563.33 mCherry 1mpU 140 24 5273213.33 mCherry 5mC/1mpU 160 245627163.33 mCherry 1mpU 160 24 4876160

Example 45 Modified Nucleic Acids with a mir-122 Sequence

A. HeLa Cells

HeLa cells were seeded at a density of 15,000 per well in 100 ul cellculture medium (DMEM+10% FBS). G-CSF mRNA having a miR-122 sequence inthe 3′UTR (G-CSF miR122; mRNA sequence shown in SEQ ID NO: 5024; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytidine and 1-methylpseudouridine) orG-CSF mRNA having a miR-122 sequence without the seed sequence in the3′UTR (G-CSF seedless; mRNA sequence shown in SEQ ID NO: 5028; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytidine and 1-methylpseudouridine)were transfected with 0.3 ul per well of Lipofectamine 2000 at aconcentration of 75 ng of mRNA per well in 96 well plates. Thesupernatant was collected between 16-18 hours after transfection andexpression of G-CSF was measured by ELISA, and the results are shown inTable 46.

TABLE 46 G-CSF Expression in HeLa Protein Expression Description (ng/ml)G-CSF 292.1 miR122 G-CSF 335.7 seedless

B. Primary Human and Rat Hepatocytes

Primary human or rat hepatocytes cells were seeded at a density of350,000 cells per well in 500 ul cell culture medium (InvitroGRO CP andInVitroGRO HI Medium+2.2% Torpedo Antibiotic Mix). G-CSF mRNA having amiR-122 sequence in the 3′UTR (G-CSF miR122; mRNA sequence shown in SEQID NO: 5024; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine) or G-CSF mRNA having a miR-122 sequence withoutthe seed sequence in the 3′UTR (G-CSF seedless; mRNA sequence shown inSEQ ID NO: 5028; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine) were transfected with 1 ul per well ofLipofectamine 2000 at a concentration of 500 ng of mRNA per well in 24well plates for the primary human hepatocytes and the primary rathepatocytes. The supernatant was collected between 16-18 hours aftertransfection and expression of G-CSF was measured by ELISA, and theresults are shown in Table 47. The mir-122 binding site sequence in themRNA dampened the G-CSF protein expression in the primary hepatocytes.

TABLE 47 G-CSF Expression in Hepatocytes Primary Human HepatocytesPrimary Rat Hepatocytes Description Protein Expression (ng/ml) ProteinExpression (ng/ml) G-CSF 116 26 miR122 G-CSF 463 85 seedless

Example 46 Time Course of Modified Nucleic Acids with a mir-122 Sequence

A. HeLa Cells

HeLa cells were seeded at a density of 17,000 per well in 100 ul cellculture medium (DMEM+10% FBS). G-CSF mRNA without a miR-122 sequence inthe 3′UTR (G-CSF; mouse 3′ UTR mRNA sequence shown in SEQ ID NO: 5717;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5716; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA having a miR-122 sequence in the3′UTR (G-CSF miR122; mouse 3′ UTR mRNA sequence shown in SEQ ID NO:5024; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5018; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA having a miR-122 seed sequence in the3′UTR (G-CSF seed; mouse 3′ UTR mRNA sequence shown in SEQ ID NO: 5026;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5020; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA having a miR-122 sequence without theseed sequence in the 3′UTR (G-CSF seedless; mouse 3′ UTR mRNA sequenceshown in SEQ ID NO: 5028; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine; human 3′UTR mRNA sequenceshown in SEQ ID NO: 5022; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine), Factor IX mRNA without amiR-122 sequence in the 3′UTR (FIX; mouse 3′ UTR mRNA sequence shown inSEQ ID NO: 5719; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5718; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), Factor IX mRNA having a miR-122 sequence in the3′UTR (FIX miR122; mouse 3′ UTR mRNA sequence shown in SEQ ID NO: 5036;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5030; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), Factor IX mRNA having a miR-122 seed sequence inthe 3′UTR (FIX seed; mouse 3′ UTR mRNA sequence shown in SEQ ID NO:5038; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5032; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine) or Factor IX mRNA having a miR-122 sequencewithout the seed sequence in the 3′UTR (FIX seedless; mouse 3′ UTR mRNAsequence shown in SEQ ID NO: 5040; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine; human 3′UTR mRNA sequenceshown in SEQ ID NO: 5034; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine) were transfected with 0.3 ulper well of Lipofectamine 2000 at a concentration of 75 ng of mRNA perwell in 96 well plates. The supernatant was collected between 16-18hours after transfection, expression of G-CSF or Factor IX was measuredby ELISA, and the results are shown in Table 48.

TABLE 48 Expression in HeLa Protein Expression Mm Protein Expression3′UTR Hs 3′UTR Description (ng/ml) (ng/ml) G-CSF 271.72 69.4 G-CSF305.36 68.8 miR122 G-CSF seed 209.5 98.0 G-CSF 243.2 80.9 seedless FIX249.8 131.6 FIX mir122 204.6 55.4 FIX seed 290.05 127.6 FIX seedless180.9 31.6

B. Primary Human and Rat Hepatocytes

Primary human or rat hepatocytes cells were seeded at a density of350,000 cells per well in 500 ul cell culture medium (InvitroGRO CP andInVitroGRO HI Medium+2.2% Torpedo Antibiotic). G-CSF mRNA without amiR-122 sequence in the 3′UTR (G-CSF; mouse 3′ UTR mRNA sequence shownin SEQ ID NO: 5717; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytidineand 1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5716; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA having a miR-122 sequence in the3′UTR (G-CSF miR122; mouse 3′ UTR mRNA sequence shown in SEQ ID NO:5024; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5018; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA having a miR-122 seed sequence in the3′UTR (G-CSF seed; mouse 3′ UTR mRNA sequence shown in SEQ ID NO: 5026;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5020; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA having a miR-122 sequence without theseed sequence in the 3′UTR (G-CSF seedless; mouse 3′ UTR mRNA sequenceshown in SEQ ID NO: 5028; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine; human 3′UTR mRNA sequenceshown in SEQ ID NO: 5022; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine), Factor IX mRNA without amiR-122 sequence in the 3′UTR (FIX; mouse 3′ UTR mRNA sequence shown inSEQ ID NO: 5719; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5718; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), Factor IX mRNA having a miR-122 sequence in the3′UTR (FIX miR122; mouse 3′ UTR mRNA sequence shown in SEQ ID NO: 5036;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5030; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), Factor IX mRNA having a miR-122 seed sequence inthe 3′UTR (FIX seed; mouse 3′ UTR mRNA sequence shown in SEQ ID NO:5038; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine; human 3′UTR mRNA sequence shown in SEQ ID NO:5032; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine) or Factor IX mRNA having a miR-122 sequencewithout the seed sequence in the 3′UTR (FIX seedless; mouse 3′ UTR mRNAsequence shown in SEQ ID NO: 5040; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine; human 3′UTR mRNA sequenceshown in SEQ ID NO: 5034; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine) were transfected with 1 ulper well of Lipofectamine 2000 at a concentration of 500 ng per well in24 well plates for the primary human hepatocytes and the primary rathepatocytes. The supernatant was collected at 24 hours, 48 hours and 72hours after transfection, expression of G-CSF and Factor IX was measuredby ELISA, and the results are shown in Table 49. The mir-122 bindingsite sequence in the mRNA dampened the G-CSF and Factor IX proteinexpression in the primary hepatocytes.

TABLE 49 G-CSF Expression in Hepatocytes Primary Human Primary HumanHepatocytes Hepatocytes Protein Expression Protein Expression (ng/ml)(ng/ml) Description Time Point Mm 3′UTR Hs 3′UTR G-CSF 24 hours 43.984.9 48 hours 18.8 100.4 72 hours 5.7 21.3 G-CSF miR122 24 hours 6.924.0 48 hours .7 3.03 72 hours .12 .88 G-CSF seed 24 hours 48.5 115.8 48hours 25.6 96.4 72 hours 8.2 19.2 G-CSF seedless 24 hours 31.7 113.1 48hours 11.7 92.9 72 hours 3.4 18.9 FIX 24 hours 90.8 63.2 48 hours 159.6124.8 72 hours 70.5 44.3 FIX mir122 24 hours 11.8 15.9 48 hours 5.0 4.472 hours 1.0 .4 FIX seed 24 hours 77.2 60.2 48 hours 115.0 63.0 72 hours41.7 20.1 FIX seedless 24 hours 69.3 53.7 48 hours 123.8 75.0 72 hours49.0 24.5

Example 47 Time Course of Modified Nucleic Acids with a Mir-122 Sequencein Cancer Cells

A. Base Level of miR-122

The base level of mir-122 in Human hepatocytes, rat hepatocytes, humanhepatocellular carcinoma cells (Hep3B) and HeLa cells were determined byTAQMAN® analysis using the manufacturers protocol. The levels werenormalized to U6 and the results are shown in Table 50.

TABLE 50 miR-122 Levels in Various Cell Types miR-122 level (normalizedto Cell Type U6) Human 16.8 Hepatocytes Rat Hepatocytes 10.9 Hep3B 0HeLa 0

B. Primary Human Hepatocytes and Hep3B Cells

Primary human hepatocytes were seeded at a density of 50,000 cells perwell in 100 ul cell culture medium (InvitroGRO CP and InVitroGRO HIMedium+2.2% Torpedo Antibiotic Mix) and Hep3B cells were seeded at adensity of 20,000 cells per well in 100 ul cell culture medium MEM+10%FBS. G-CSF mRNA without a miR-122 sequence in the 3′UTR (G-CSF; mRNAsequence shown in SEQ ID NO: 5745; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, cap1; fully modified with5-methylcytidine and 1-methylpseudouridine), G-CSF mRNA having a miR-122sequence in the 3′UTR (G-CSF miR122; mRNA sequence shown in SEQ ID NO:5018; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA having a miR-122 seed sequence in the3′UTR (G-CSF seed; mRNA sequence shown in SEQ ID NO: 5020; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, cap1; fullymodified with 5-methylcytidine and 1-methylpseudouridine) or G-CSF mRNAhaving a miR-122 sequence without the seed sequence in the 3′UTR (G-CSFseedless; mRNA sequence shown in SEQ ID NO: 5022; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytidine and 1-methylpseudouridine) weretransfected with 0.3 ul per well of Lipofectamine 2000 at aconcentration of 75 ng of mRNA per well in 96 well plates for theprimary human hepatocytes and the Hep3B cells. The supernatant wascollected at 24 hours, 48 hours and 72 hours after transfection,expression of G-CSF was measured by ELISA, and the results are shown inTable 51. The mir-122 binding site sequence in the mRNA dampened theG-CSF protein expression in the primary human hepatocytes but not in theHep3B cells.

TABLE 51 G-CSF Expression Primary Human Hepatocytes Hep3B ProteinExpression Protein Expression (ng/ml) (ng/ml) Description Time Point Hs3′UTR Hs 3′UTR G-CSF 24 hours 76 55 48 hours 12 33 72 hours 6 10 G-CSFmiR 122 24 hours 32 37 48 hours 1 27 72 hours 0 6 G-CSF seed 24 hours 7539 48 hours 11 28 72 hours 4 6 G-CSF seedless 24 hours 79 49 48 hours 1535 72 hours 6 9

Example 48 Time Course of Modified Nucleic Acids with a mir-142 3pSequence

A. Base Level of miR-143 3p

The base level of miR-142 3p in RAW264.7 cells and HeLa cells weredetermined by TAQMAN® analysis using the manufacturer's protocol. Thelevels were normalized to U6 and the results are shown in Table 52.

TABLE 52 miR-142 3p Levels in Various Cell Types miR-122 level Cell Type(normalized to U6) Human 16.8 Hepatocytes Rat Hepatocytes 10.9 Hep3B 0HeLa 0

B. HeLa and RAW264.7 Cells

HeLa cells were seeded at a density of 17,000 per well in 100 ul cellculture medium DMEM+10% FBS and RAW264.7 cells were seeded at a densityof 200,000 per well in 100 ul cell culture medium DMEM+10% FBS. G-CSFmRNA without a miR-142 3p sequence in the 3′UTR (G-CSF; mRNA sequenceshown in SEQ ID NO: 5749; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine), G-CSF mRNA having a miR-1423p sequence in the 3′UTR (G-CSF miR142 3p; mRNA sequence shown in SEQ IDNO: 5750; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA having a miR-142 3p seed sequence inthe 3′UTR (G-CSF seed; mRNA sequence shown in SEQ ID NO: 5751; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,cap1; fully modified with 5-methylcytidine and 1-methylpseudouridine) orG-CSF mRNA having a miR-142 3p sequence without the seed sequence in the3′UTR (G-CSF seedless; mRNA sequence shown in SEQ ID NO: 5752; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytidine and 1-methylpseudouridine)were transfected with 0.3 ul per well of Lipofectamine 2000 at aconcentration of 75 ng of mRNA per well in 96 well plates for HeLa orwith 1 ul per well of Lipofectamine 2000 at a concentration of 250 ng ofmRNA per well in 24 well plates for RAW264.7 cells. The supernatant wascollected 16-18 hours after transfection, expression of G-CSF wasmeasured by ELISA, and the results are shown in Table 53. miR-142 3psites in G-CSF were shown to down-regulate G-CSF expression in RAW264.7cells.

TABLE 53 Expression HeLa RAW264.7 Protein Expression Protein ExpressionDescription (ng/ml) (ng/ml) G-CSF 243.5 124.8 G-CSF miR 142 309.1 42.83p G-CSF seed 259.8 148.1 G-CSF seedless 321.7 185.2

C. Time Course in RAW264.7 Cells

RAW264.7 cells were seeded at a density of 60,000 cells per well in 100ul cell culture medium (DMEM+10% FBS). G-CSF mRNA without a miR-142 3psequence in the 3′UTR (G-CSF; mRNA sequence shown in SEQ ID NO: 5749;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA having a miR-142 3p sequence in the3′UTR (G-CSF miR142 3p; mRNA sequence shown in SEQ ID NO: 5750; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytidine and 1-methylpseudouridine),G-CSF mRNA having a miR-142 3p seed sequence in the 3′UTR (G-CSF seed;mRNA sequence shown in SEQ ID NO: 5751; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, cap1; fully modified with5-methylcytidine and 1-methylpseudouridine) or G-CSF mRNA having amiR-142 3p sequence without the seed sequence in the 3′UTR (G-CSFseedless; mRNA sequence shown in SEQ ID NO: 5752; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, cap1; fullymodified with 5-methylcytidine and 1-methylpseudouridine) weretransfected with 0.3 ul per well of Lipofectamine 2000 at aconcentration of 75 ng of mRNA per well in 96 well plates. Thesupernatant was collected at 24 hours, 48 hours and 72 hours aftertransfection, expression of G-CSF was measured by ELISA, and the resultsare shown in Table 54. The mir-142 3p binding site sequence in the mRNAshowed a strong suppression of G-CSF expression in RAW264.7 cells overtime.

TABLE 54 G-CSF Expression RAW264.7 Cells Description Time Point ProteinExpression (ng/ml) G-CSF 24 hours 133.5 48 hours 69.7 72 hours 2.1 G-CSFmiR 142 3p 24 hours 60.1 48 hours 9.2 72 hours .3 G-CSF seed 24 hours244.9 48 hours 68.9 72 hours 2.3 G-CSF seedless 24 hours 250.2 48 hours95.9 72 hours 3.0

D. miR-142 3p in PBMC

Peripheral blood mononuclear cells (PBMCs) were seeded at a density of150,000 cells per well in 100 ul cell culture medium (Opti-MEM and aftertransfection add 10% FBS). G-CSF mRNA having a miR-142 3p sequence inthe 3′UTR (G-CSF miR142 3p; mRNA sequence shown in SEQ ID NO: 5750;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA having a miR-142 3p seed sequence inthe 3′UTR (G-CSF seed; mRNA sequence shown in SEQ ID NO: 5751; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,cap1; fully modified with 5-methylcytidine and 1-methylpseudouridine) orG-CSF mRNA having a miR-142 3p sequence without the seed sequence in the3′UTR (G-CSF seedless; mRNA sequence shown in SEQ ID NO: 5752; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,cap1; fully modified with 5-methylcytidine and 1-methylpseudouridine)were transfected in triplicate with 0.4 ul per well of Lipofectamine2000 at a concentration of 500 ng of mRNA per well in 96 well plates for2 or 3 donors. The supernatant was collected at 24 hours aftertransfection and the expression of G-CSF was measured by ELISA. Theresults for the 2 donors are shown in Table 55 and the results for the 3donors are shown in Table 56. The mir-142 3p binding site sequence inthe mRNA was shown to down regulate G-CSF expression in human PBMC.

TABLE 55 Expression PBMC (2 donors) Description Protein Expression(ng/ml) G-CSF miR 142 3p 5.09 G-CSF seed 10.06 G-CSF seedless 9.38

TABLE 56 Expression PBMC (3 donors) Description Protein Expression(ng/ml) G-CSF miR 142 3p 7.48 G-CSF seed 13.40 G-CSF seedless 13.98

Example 49 In Vivo Expression of Modified mRNA

A. BALB/C Nude Mice

BALB/c nude mice were injected intravenously with 0.1 mg/kg luciferasemodified mRNA without a miR-122 binding site (“non-targeted mRNA”; mRNAsequence shown in SEQ ID NO: 5753; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpeudouridine) formulated in a lipidnanoparticle described in Table 57 or luciferase modified mRNA with amiR-122 binding site in the 3′UTR (“miR-122 targeted mRNA”; mRNAsequence shown in SEQ ID NO: 5754; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpeudouridine) formulated in a lipidnanoparticle described in Table 58.

TABLE 57 Lipid Nanoparticle for Non-targeted mRNA LNP Luciferase:non-targeted mRNA Lipid DLin-KC2-DMA Lipid/RNA wt/wt 20 Mean size 73.3nm PDI: 0.06

TABLE 58 Lipid Nanoparticle for Targeted mRNA LNP Luciferase: targetedmRNA Lipid DLin-KC2-DMA Lipid/RNA wt/wt 20 Mean size 70.6 nm PDI: 0.08

24 hours post-treatment, animals were anesthetized, injected with theluciferase substrate D-luciferin and the bioluminescence imaging (BLI)from living animals was evaluated in an IVIS imager 15 minutes later.Signals were obtained from animals injected with non-targeted mRNA andfrom miR-122 targeted mRNA, and presented in Table 59. The total lightsignal produced from livers of animals treated with miR 122 targetedmRNA is 29× lower than non-targeted mRNA, showing that the engineeredelement in the 3′UTR may inhibit protein expression in normal tissue.

TABLE 59 In vivo expression of modified mRNA modulated by an engineeredmiR 122 binding site Luciferase signal from liver Description(photons/sec) Non-targeted mRNA 7.9 × 10⁷ miR-122 targted mRNA 2.7 × 10⁶

B. BALB/c Nude Mice with Hepatocellular Carcinoma Hep3B Cells

BALB/c nude mice were intrahepatically implanted with 2×10⁶hepatocellular carcinoma Hep3B cells and resulting orthotopic tumorsallowed to grow for 24 days. Tumor-bearing mice were then intravenouslyinjected with 0.1 mg/kg luciferase modified mRNA without a miR-122binding site (“non-targeted mRNA”; mRNA sequence shown in SEQ ID NO:5753; polyA tail of approximately 140 nucleotides not shown in sequence;5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpeudouridine) or luciferase modified mRNA with a miR-122 bindingsite in the 3′UTR (“miR-122 targeted mRNA”; mRNA sequence shown in SEQID NO: 5754; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpeudouridine) formulated in a lipid nanoparticle described inTable 57 and 58 (above). 24 hr post-treatment animals were anesthetized,injected with the luciferase substrate D-luciferin and bioluminescenceimaging (BLI) from living animals was evaluated in an IVIS imager 20minutes later. Signal from orthotopic tumors compared to adjacent normalliver was quantified, and miR-122-targeted mRNA systemically deliveredvia lipid nanoparticles achieved over 2-fold enrichment in tumorcompared to normal liver.

Example 50 Effect of the Kozak Sequence in Modified Nucleic Acids

HeLa cells were seeded at a density of 15,000 per well in 100 ul cellculture medium DMEM+FBS 10%. G-CSF mRNA having a Kozak sequence (G-CSFKozak; mRNA sequence shown in SEQ ID NO: 5716; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytidine and 1-methylpseudouridine), G-CSF mRNAnot having a Kozak sequence (G-CSF no Kozak; mRNA sequence shown in SEQID NO: 5008; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytidine and1-methylpseudouridine), G-CSF mRNA where the −3 position A, upstream ofthe start codon, was converted to a T, (G-CSF 3t5′; mRNA sequence shownin SEQ ID NO: 5755 (Table 60); polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytidine and 1-methylpseudouridine), G-CSF mRNA where the −9position A, upstream of the start codon, was converted to a T, (G-CSF9t5′; mRNA sequence shown in SEQ ID NO: 5756 (Table 60); polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytidine and 1-methylpseudouridine) or G-CSF mRNAwhere 9 nucleotides upstream of the start codon (AGAGCCACC) were deleted(G-CSF 9de15′; mRNA sequence shown in SEQ ID NO: 5757 (Table 60); polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytidine and 1-methylpseudouridine)and transfected in triplicate at a concentration of 37.5 ng per well in96 well plates. 24 hours, 48 hours and 72 hours after transfection, thesupernatant was collected and expression of G-CSF was measured by ELISA,and the results are shown in Table 61. In Table 60, the start codon ineach sequence is underlined. In Table 60, for G-CSF 3t5′ the −3 positionA, upstream of the start codon is in bold and underlined and for G-CSF9t5′ the −9 position A upstream of the start codon is in bold andunderlined.

TABLE 60 G-CSF Sequences SEQ De- ID scription Sequence NO G-CSFGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAA 5755 3t5′ UAUAAGAGCC UCCAUGGCCGGUCCCGCGACCCAAA GCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCG ACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAG AUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGA GCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUU GCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCU UGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAA CAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGC CGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUU UUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGGAGCCUCGGUGGC CAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUU UGAAUAAAGUCUGAGUGGGCGGC G-CSFGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAA 5756 9t5′ UAUA UGAGCCACCAUGGCCGGUCCCGCGACCCAAA GCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCG ACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAG AUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGA GCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUU GCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCU UGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAA CAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGC CGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUU UUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGGAGCCUCGGUGGC CAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUU UGAAUAAAGUCUGAGUGGGCGGC G-CSFGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAA 5757 9del5′UAUAAUGGCCGGUCCCGCGACCCAAAGCCCCAUGA AACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCG GACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGC GAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACU GCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGG CAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAA UCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUC UGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUU GCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGU CUCGUACCGGGUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUC UUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAA AGUCUGAGUGGGCGGC

TABLE 61 G-CSF Expression 24 hours 48 hours 72 hours Protein ProteinProtein Expression Expression Expression (ng/ml) (ng/ml) (ng/ml) G-CSFKozak 239.08 339.89 283.43 G-CSF No Kozak 399.83 544.08 437.23 G-CSF3t5′ 157.39 239.67 195.20 G-CSF 9t5′ 171.84 263.11 195.22 G-CSF 9del5′308.16 563.64 397.20

Example 51 Effect of Modification of 5′UTR in Modified Nucleic Acids

BJ Fibroblast cells were seeded at a density of 100,000 per well in 500ul cell culture medium EMEM+FBS 10%. G-CSF mRNA having a synthetic 5′UTR(G-CSF; mRNA sequence shown in SEQ ID NO: 5716; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytidine and 1-methylpseudouridine) or G-CSF mRNAcontaining a 5′UTR with five tandem repeats of an 18 nucleotide sequencefrom the IRES of the GTX gene (GTX G-CSF; mRNA sequence shown in SEQ IDNO: 5758 (Table 62); polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytidineand 1-methylpseudouridine) and transfected in triplicate at aconcentration of 250 ng per well in 24 well plates. 24 hours, 48 hoursand 72 hours after transfection, the supernatant was collected andexpression of G-CSF was measured by ELISA, and the results are shown inTable 63. In Table 62, the start codon is underlined and the five tandemrepeats of an 18 nucleotide sequence from the IRES of the GTX gene isbolded and the first, third and fifth tandem repeat of the 18 nucleotidesequence is also underlined.

TABLE 62 GTX G-CSF Sequence SEQ De- ID scription Sequence NO GTX G- GGGAAAUUCUGACAUCCGGCGG AAUUCUGACAU 5758 CSF CCGGCGG AAUUCUGACAUCCGGCGGAAUUCUGA CAUCCGGCGG AAUUCUGACAUCCGGCGG AAGACUCACAACCCCAGAAACAGACAUUAAGAGAGAAAA GAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGG CCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUC AUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCG CACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCAC AGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCU UUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAG AAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAG AUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCG UUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCG GGUGCUGAGACAUCUUGCGCAGCCGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUU GGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGU GGGCGGC

TABLE 63 5′ UTR G-CSF Gtx G-CSF Protein Protein Expression ExpressionTime point (ng/ml) (ng/ml) 24 hours 26.13 79.65 48 hours 138.75 444.8172 hours 55.37 198.14

Example 53 Effect of Modification of 5′UTR in Modified Nucleic Acids

BJ Fibroblast cells were seeded at a density of 100,000 per well in 500ul cell culture medium EMEM+FBS 10%. G-CSF mRNA having a synthetic 5′UTR(G-CSF; mRNA sequence shown in SEQ ID NO: 5716; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 1-methylpseudouridine) or G-CSF mRNA containing a 5′UTRwith five tandem repeats of an 18 nucleotide sequence from the IRES ofGtx gene (Gtx G-CSF; mRNA sequence shown in SEQ ID NO: 5758 (Table 62);polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 1-methylpseudouridine) and transfected intriplicate at a concentration of 250 ng per well in 24 well plates. 24hours, 48 hours and 72 hours after transfection, the supernatant wascollected and expression of G-CSF was measured by ELISA, and the resultsare shown in Table 64.

TABLE 64 5′ UTR G-CSF Gtx G-CSF Protein Protein Expression ExpressionTime point (ng/ml) (ng/ml) 24 hours 129.10 178.68 48 hours 569.971067.62 72 hours 325.16 738.30

Example 54 In Vivo Effect of the Modification of 5′UTR in Nucleic AcidsModified with 5-methylcytidine and 1-methylpseudouridine

To study the effect of the modification of the 5′UTR in modified nucleicacids female Balb/c mice (n=3; 12 weeks old; Harlan Laboratories (SouthEaston, Mass.)) were treated with lipoplexed mRNA.

8 ug of G-CSF mRNA having a synthetic 5′UTR (G-CSF; mRNA sequence shownin SEQ ID NO: 5716; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytidineand 1-methylpseudouridine) or G-CSF mRNA where 9 nucleotides upstream ofthe start codon were deleted (G-CSF 9de15′; mRNA sequence shown in SEQID NO: 5757 (Table 60); polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytidineand 1-methylpseudouridine) for treatment of 3 mice is diluted withsterile and serum-free DMEM (Life Technologies) to obtain a total volumeof 200 ul. A total of 8 ul of Lipofectamine-2000 (LifeTechnologies,11668019) for the treatment of 3 mice was diluted with sterile andserum-free DMEM (LifeTechnologies, Grand Island, N.Y.; 11965-118) toobtain a total volume of 200 ul. After 5 minutes of incubation, the twosolutions were combined and carefully mixed with a pipette. After 20minutes the formation of mRNA-Lipofectamine-2000 lipoplexes wascompleted. The lipoplex solution was transferred to a sterile 1 mlsyringe (BD Falcon) carrying a 27 gauge injection needle (0.3 mL BDSafetyGlide insulin syringe with 29 G×½ in BD permanently attachedneedle (Catalog #305935)). The Balb/C mice were placed under a heat lampfor 5 minutes prior to the 100 ul intravenous tail vein injectioncontaining 2 ug of lipoplexed mRNA. 6 hours after injection the micewere anesthesized and bleed for serum collection by cardiac puncture.The serum samples were then run on a G-CSF ELISA (R&D systems catalog#SCS50) and the results are shown in Table 65. The G-CSF mRNA having the9 nucleotides upstream of the start codon deleted had a higher G-CSFexpression level at 6 hours as compared to the G-CSF having a syntheticUTR.

TABLE 65 G-CSF Kozak Expression In Vivo G-CSF G-CSF 9del5′ Time PointExpression (ng/ml) Expression (ng/ml) 6 hours 256.2 752.4

Example 55 In Vivo Effect of the GTX Modification of 5′UTR in G-CSFNucleic Acids Modified with 5-methylcytidine and 1-methylpseudouridine

To study the effect of the modification of the 5′UTR in modified nucleicacids female Balb/c mice (n=3; 12 weeks old; Harlan Laboratories (SouthEaston, Mass.)) were treated with lipoplexed mRNA.

8 ug of G-CSF mRNA having a synthetic 5′UTR (G-CSF; mRNA sequence shownin SEQ ID NO: 5716; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytidineand 1-methylpseudouridine) or G-CSF mRNA containing a 5′UTR with fivetandem repeats of an 18 nucleotide sequence from the IRES of GTX gene(GTX G-CSF; mRNA sequence shown in SEQ ID NO: 5758 (Table 62); polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytidine and 1-methylpseudouridine)for treatment of 3 mice is diluted with sterile and serum-free DMEM(Life Technologies) to obtain a total volume of 200 ul. A total of 8 ulof Lipofectamine-2000 (LifeTechnologies, 11668019) for the treatment of3 mice was diluted with sterile and serum-free DMEM (LifeTechnologies,Grand Island, N.Y.; 11965-118) to obtain a total volume of 200 ul. After5 minutes of incubation, the two solutions were combined and carefullymixed with a pipette. After 20 minutes the formation ofmRNA-Lipofectamine-2000 lipoplexes was completed. The lipoplex solutionwas transferred to a sterile 1 ml syringe (BD Falcon) carrying a 27gauge injection needle (0.3 mL BD SafetyGlide insulin syringe with 29G×½ in BD permanently attached needle (Catalog #305935)). The Balb/Cmice were placed under a heat lamp for 5 minutes prior to the 100 ulintravenous tail vein injection containing 2 ug of lipoplexed mRNA. 6hours after injection the mice were anesthesized and bleed for serumcollection by cardiac puncture. The serum samples were then run on aG-CSF ELISA (R&D systems catalog #SCS50) and the results are shown inTable 66. The G-CSF mRNA having five tandem repeats of an 18 nucleotidesequence from the IRES of GTX gene had a higher G-CSF expression levelat 6 hours as compared to the G-CSF having a synthetic UTR.

TABLE 66 GTX G-CSF Expression In Vivo G-CSF GTX G-CSF Time PointExpression (ng/ml) Expression (ng/ml) 6 hours 266.4 1284.4

Example 56 In Vivo Effect of the GTX Modification of 5′UTR in G-CSFNucleic Acids Modified with 1-Methylpseudouridine

To study the effect of the modification of the 5′UTR in modified nucleicacids female Balb/c mice (n=3; 12 weeks old; Harlan Laboratories (SouthEaston, Mass.)) were treated with lipoplexed mRNA.

8 ug of G-CSF mRNA having a synthetic 5′UTR (G-CSF; mRNA sequence shownin SEQ ID NO: 5716; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with1-methylpseudouridine) or G-CSF mRNA containing a 5′UTR with five tandemrepeats of an 18 nucleotide sequence from the IRES of GTX gene (GTXG-CSF; mRNA sequence shown in SEQ ID NO: 5758 (Table 62); polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 1-methylpseudouridine) for treatment of 3 mice is dilutedwith sterile and serum-free DMEM (Life Technologies) to obtain a totalvolume of 200 ul. A total of 8 ul of Lipofectamine-2000(LifeTechnologies, 11668019) for the treatment of 3 mice was dilutedwith sterile and serum-free DMEM (LifeTechnologies, Grand Island, N.Y.;11965-118) to obtain a total volume of 200 ul. After 5 minutes ofincubation, the two solutions were combined and carefully mixed with apipette. After 20 minutes the formation of mRNA-Lipofectamine-2000lipoplexes was completed. The lipoplex solution was transferred to asterile 1 ml syringe (BD Falcon) carrying a 27 gauge injection needle(0.3 mL BD SafetyGlide insulin syringe with 29 G×½ in BD permanentlyattached needle (Catalog #305935)). The Balb/C mice were placed under aheat lamp for 5 minutes prior to the 100 ul intravenous tail veininjection containing 2 ug of lipoplexed mRNA. 6 hours after injectionthe mice were anesthesized and bleed for serum collection by cardiacpuncture. The serum samples were then run on a G-CSF ELISA (R&D systemscatalog #SCS50) and the results are shown in Table 67. The G-CSF mRNAhaving five tandem repeats of an 18 nucleotide sequence from the IRES ofGTX gene had a higher G-CSF expression level at 6 hours as compared tothe G-CSF having a synthetic UTR.

TABLE 67 GTX G-CSF Expression In Vivo G-CSF GTX G-CSF Time PointExpression (ng/ml) Expression (ng/ml) 6 hours 5638.2 6281.1

Other Embodiments

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, databases, databaseentries, other references and art mentioned herein are incorporated byreference in their entirety, even if not expressly stated in thecitation. In case of conflict, the present specification, includingdefinitions, will control. In addition, section headings, the materials,methods, and examples are illustrative only and not intended to belimiting.

The invention claimed is:
 1. A method of reducing a proteinantigen-mediated immune response in a subject, comprising administeringto the subject a composition comprising a chemically modified mRNA and apharmaceutically acceptable carrier, wherein the chemically modifiedmRNA comprises (a) a 5′ untranslated region (5′ UTR); (b) a region oflinked nucleosides encoding said protein antigen; (c) a 3′ untranslatedregion (3′ UTR) comprising at least one miR-142-3p microRNA bindingsite; and (d) a 3′ tailing region of linked nucleosides, wherein thechemically modified mRNA comprises fully modified 1-methyl pseudouridinenucleosides, and optionally comprises fully modified 5′ methyl-cytidinenucleosides, thereby reducing the protein antigen-mediated immuneresponse in the subject.
 2. The method of claim 1, wherein the firstregion of linked nucleosides is codon optimized.
 3. The method of claim1, wherein the chemically modified mRNA comprises fully modified1-methyl pseudouridine nucleosides without fully modified 5′methyl-cytidine nucleosides.
 4. The method of claim 1, wherein thechemically modified mRNA comprises fully modified 1-methyl pseudouridinenucleosides and fully modified 5′ methyl-cytidine nucleosides.
 5. Themethod of claim 1, wherein the miR-142-3p microRNA binding sitecomprises the sequence set forth in SEQ ID NO:
 1404. 6. The method ofclaim 1, wherein the 3′ tailing region of linked nucleosides comprises apoly A tail of at least 100, at least 120, or at least 140 nucleosides.7. The method of claim 1, wherein the chemically modified mRNA furthercomprises a 5′ cap structure.
 8. The method of claim 7, wherein the 5′cap structure comprises Cap1.
 9. The method of claim 1, wherein theprotein antigen is a therapeutic protein, cytokine, growth factor,antibody or fusion protein.
 10. The method of claim 1, wherein thechemically modified mRNA is formulated with a liposome, lipoplex, orlipid nanoparticle.
 11. The method of claim 10, wherein the chemicallymodified mRNA is formulated with a lipid nanoparticle.
 12. The method ofclaim 11, wherein the lipid nanoparticle comprises a cationic orionizable lipid.
 13. The method of claim 12, wherein the cationic lipidis selected from the group consisting of DLin-MC3-DMA, DLin-DMA, C12-200and DLin-KC2-DMA.